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|261 m²
|Tipo de imóvel
|Estadia Mínima
|3 - 7 noites
Umas férias no The View é uma praia de férias incomparável em alojamento, localização e beleza em qualquer lugar da costa leste !. Esta bela casa de 3 andares, com elevador e piscina é o Ultimate Experience Beach! Você vai adorar os seus dias na praia, seguido por um profundo relaxamento na piscina incrível! Três histórias de conforto com as mãos para baixo a melhor vista e localização na ilha. Esta bonita casa de férias oferece a cada alojamento para toda a família com cada quarto maravilhosamente nomeado para a beleza e conforto. Um elevador varre a cada andar, onde você e seus entes one vai criar memórias duradouras da Best Vacation Ever.
O Vista tem 6 quartos bonitos, 5 banheiros, chuveiro ao ar livre, elevador, piscina e todas as comodidades imagináveis. Você vai adorar os seus dias a nadar, pescar e brincar na areia fina seguida de noites de conforto e diversão em acomodações de luxo.
Venha para o Vista e saber a melhor experiência possível férias praia nunca!
Bad weather for our gathering unfortunately but the property and location are great and the rental/management staff were super!
After going to Ocean Isle for twelve years, we decided to go to this beautiful beach house at Emerald Isle. The house was beautifully decorated, very clean and very well equipped. My family is 6 adults and 5 children under the age of 7. The house is perfectly laid out for us. The pool is right off the family room and the beach is a stones throw from the house. The views from the house are extraordinary. The owner was wonderful to work with. We will definitely return to this peaceful beautiful home.
Our family of 15 (10 adults, 5 little kids/babies) stayed at this ocean front house. We chose it because it has 6 bedrooms, its own pool, and an elevator. It worked out so well for us. It was clean, very nicely decorated, and had all the conveniences of home. We thought the pool looked small in the photos but it was plenty big, and it had both shallow and deep areas. We liked how the pool was next to the house but you couldn't walk directly from the house to the pool, you still had the security gate to keep the little ones safe. The beds/baths are like this: ground floor has a bunkbed room + bath, and another bedroom + bath, a laundry room with a sink, and a game/TV room that has a fold out sofa that we could've used as an extra bedroom. The main floor (second level) has the living room, dining room, kitchen, plus a king bedroom + bath. The stairs are off to the side a bit so we didn't have to worry that the two year old and the crawling baby would fall down the stairs. The top floor has the master bedroom with an ensuite bathroom, plus two other bedrooms that share a bathroom. All the bathrooms in the whole house except the master have bathrooms just outside the bedrooms, not IN the bedrooms, so someone can access the bathroom without waking a late sleeper. There are several balconies with lots of seating. The balconies and many house windows offer beautiful views of the ocean and beach. There is a helpful elevator, which a few of us used. There is parking for four cars on the private lot and room for our fifth car just across the street. Beach access is very convenient and the beach itself was really nice too, we spent lots of time there. The dining room was a bit small for the house, the table sits 8 and we often pulled one or more of the counter seats over, but there was always one or more of us eating in the living room. The owners had a high chair, crib, etc all set up for us. We would highly recommend this house!
Beautiful property with easy access to the beach. A great place for gathering with family. The elevator was an especially helpful amenity for our Grandmother. We plan on returning!
We had 3 families with a total of 12 people stay in the house for our beach vacation. We really liked the area and enjoyed the house. Our experience was mostly positive with just a few minor exceptions. First the positives.
* house location worked out well. We were a little concerned with how crowded the beach might be since the house is next to a resort. We were pleasantly surprised. Our vacation started on Memorial Day weekend and the entire beach was very crowded - except for the spot in front of the few houses. There seemed to be an open space of at least several hundred yards on either side of us before the crowds started. After Memorial Day there were no crowds at all.
*We were concerned that our only beach access was a boardwalk on the other side of the house next door. We thought this was going to bed very inconvenient, but to be honest, we didn't even notice after a few trips (the nice people next door even let us sneak through their back yards sometimes to make it shorter).
*The house itself was newer with freshly painted walls and was nicely decorated. The kitchen was fully stocked (with the exception of oven mitts). The house included a nice gas grill (even had a spare tank handy) and the pool was a nice touch.
We did have a few minor issues to mention.
*The AC vent in one of the main bedrooms did not work. The management company worked diligently to find the problem and were willing to get a crew out to fix it. We decided against having any construction during our vacation though. The owner took care of the issue in a very equitable way though.
*The outdoor shower left a little to be desired. Most beach houses I've been too have either a pull out handle, a foot spray, or both. This just had a normal shower head which made it difficult to get the sand off the shorter kids.
*The house had plenty of bathrooms, but we did notice that when more than 2 or 3 people showered at the same time there was almost no water pressure.
Overall we really enjoyed our time and I would recommend staying at this house. The small issues we experienced were far outweighed by the positives.
The View fica a poucos minutos do Aquário da Carolina do Norte em Pine Knoll Shores e Emerald Isle. O Vista tem uma vista fantástica para o mar e é muito bem desenhados e decorados.
|Validade da Tarifa
|por noite
|Noite (fim de semana)
|por semana
|por mês
|Pacote ou Evento
Season 2017
mai 27 2017 - set 8 2017
Estadia mínima: 7 noite(s)
Off Season 2017
set 9 2017 - dez 31 2017
Estadia mínima: 3 noite(s)
|Cleaning fee
|Taxa de animais de estimação
|Linen fee


text:
Meet AmberMature
She nodded, "of course, it's soothing. It's almost like a lullaby; I can hear it, beating. It's louder now...I'm getting my senses back and it's so sweet," she murmured, her eyes closing as she relaxed with him.
The air began to smell sweet with the cake shop that lay just down the road. She could smell the smoke from cars as they whizzed around down below. Colours seemed brighter, more attuned and as she opened one eye she could see a woman sat in an office cubicle on the phone, in the building across from them. She had an open box of doughnuts on her desk that was beside her bag. As she put the phone down and picked it up again, Melissa even saw the number she dialled.
But then she simply closed her eyes, tuning back into the fast beating of Bane's heart. "So sweet," she repeated but he remained silent.
"So, you're the most dominant form?" The voice in the back of her head asked and she confirmed it, "Melissa... it's nice to meet you."
"And you, Amber,"
"So...love, what is it? You keep thinking about it." She explained it the same way she did with Bane and the wolf made an understanding, 'Ohh.' "I'm bursting with energy, I want my form!"
Melissa sighed, "You'll have to wait, I'm sorry." This was just answered by a short huff from Amber.
"I love you, Melissa," he whispered quietly, but it caught her off guard as she was busy talking to Amber, she heard it in the background though, so she zoned back in.
"I love you," he murmured, raising his voice a little so she could hear him better.
"Oh, I love you too, Bane," she smiled for a little moment before it fell again. "But now it's not me you have to convince."
"I know," he sighed.
She nudged his side slightly, "you'll be fine... you're charming."
"Depends if Amber goes for charming," he laughed lightly.
"I don't think I know what I go for," Amber murmured through Melissa.
"You've got plenty of time to figure it out," he smiled. Amber noticed the smile and used her host to find out it was a nice gesture.
"So you're not still angry about earlier? I didn't mean to challenge your authority," she muttered, pulling away from him instinctively, making a polite distance between them and hunched her form so she was lower than him.
"No... You're a pup. I possibly should have been more tolerant," he sighed.
"Well I'm sorry, Alpha." She bowed her head, not making eye-contact with him for fear of challenging him again.
"I forgive you," he stepped forward and licked her face, before moving back again, looking out over the city, "this time," he laughed slightly.
She faltered with a frown for a while, before making Melissa smile. She found it hard to manipulate Melissa's strings to make her do anything and for the moment she was happy she was sat down. "Thank you, Alpha. I'll make it up to you." He nodded silently, wondering what to say to the pup. "You know, I like you, Alpha," Amber announced, taking both Melissa and Bane by surprise.
"What?" She asked, looking at him questioningly.
"Nothing, I just wasn't expecting it, that's all," he smiled.
"I just said I like you, you're kind, that's all," she muttered
"I try. Clashing with an alpha as a newborn isn't a good experience, I know."
"Well, don't expect anything more, just... acquaintanceship."
"Who said I was expecting more?"
"Hey, just 'cause I ain't talking, don't mean I ain't listening," he was silent for quite a while, making Amber-Melissa's smile grew into a wide grin, "I caught you out wolfie-boy," she laughed.
He growled irritably and glared at her, "do not call me 'wolfie-boy'. I only wish to protect our humans. If you insist on finding someone else, then fine, but it will be on your conscience if you tear Melissa apart."
"Ahh, but you love her and she loves you, I either love you or no one. I'm not that cruel." Amber stated coldly and Melissa breathed a sigh of relief, "sorry, I guess I sorta got too relaxed in your company, won't happen again." She murmured mockingly before clumsily rising to her feet and bowing to him.
He looked up at her for a moment and then back up at the sky, "I have a name, I suggest you use it."
She snarled, rolled her eyes and walked a few steps away before stopping, "I suggest you bite me.
"Don't tempt me," he growled.
"Try it," she barked and grinned; as much as Melissa tried, she couldn't stop Amber from turning and motioning him to bring it on.
He leapt at her, bowling her over. They crashed to the floor together, all his weight focused on her shoulders as he pinned her down with his huge paws. His teeth snapped together, clamping around her throat threateningly.
Melissa's stomach convulsed and she screamed out in agony, "Go on then, do it!" Amber managed to snarl through it all.
His jaw tightened ever so slightly, but the cry in the back of his head as Lazarus shouted at him to stop made him pause. He released her after a moment, but remained standing on her.
Melissa cried out once more, tears streaming down her face but Amber was still in control, "You wimped out," she laughed slightly, as her form began gasping for air.
"I didn't wimp out," he growled, "I'm protecting our humans."
"That's why... why she's in so much..." Amber growled as Melissa tried to fight back, "pain."
He stepped off her and walked to the other side of the roof away from her, apologising to Lazarus for snapping all the way.
"How do I fight it?" Melissa screamed, "How do I stop it?"
"You're supposed to be the dominant being. Make her listen to you," he called.
"I've never been a dominant being in my whole life."
"Well you're gonna have to be, or she'll take over," he snarled, "And make all our lives a misery at this rate," he added in a quiet mutter.
"Don't snarl at me," she commanded, bringing Amber's fury with her own. "Don't you dare snarl at me!"
"Or what?" he snapped.
Her body convulsed again but she didn't fight back this time, she was with Amber... she didn't want to back down. But the pain she felt made her tighten herself into a ball, her arms hugging around her form as her muscles grew. "Just you wait," they both chimed together.
Amber's energy and excitement about gaining her form rippled through Melissa and Melissa tried her best to communicate with her. "You do realise I'm the dominant one between us both, right?"
Amber sighed, "Yes, but I don't know how you could like this mongrel, let alone love him! I'll teach him a lesson and then you can have your body back." He turned and watched, moving away from the edge of the roof.
Melissa screamed as the pain racked her body, twisting her muscles, tearing her flesh. Her clothes were already torn and she had begun to sprout thick, golden brown fur all over her body apart from her underside where it faded to a light blonde. Her emerald green eyes dilated and tears rolled down her face as everything went numb and the transformation began to end. Her ears twitched and her tail swung like a pendulum behind her.
She opened her jaw and closed it multiple times as she adjusted and her claws scraped against the roof before she let out one long, ear-piercing howl.

subset: commoncrawl


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Crusader ~ Chapter II Part IIIMature
~ ~
The next day we decided to stay and make a camp where we were, and wait a week or two before we went to Menzoberran, so the Lokotian army could push back the Orcs. About four days in, however, we had a slight change in mind.
On the fourth day, I was going out to get some decent wood for mine and Alexander's shelter, since it rained the night before. The cool, valley mist filled the air, coating everything it touched with dew. The occasional bird chirping and the sounds of my footsteps were all I could hear for a while.
But of course, my quest for some alone time met with failure as Alexander came and followed me, his stolen chain armor rattling on.
"-Hey, wait up!"
The memories of Derynn were still fresh, and I wanted to distance myself from anything having to do with it. But at that point, that meant everything.
"... You scared away the birds."
Alexander looked up at the trees, as if he never heard them to begin with. He paused, then looked back at me.
"Katherin sent me to get some herbs- Oh! And she told me to tell you that she found where she put her spellbook yesterday."
"Her book must be ruined... What spells did she have in there?"
"Not much. Just a few cantrips, really."
"Nothing to put Derynn back the way it was?"
Alexander chuckled a bit. "No, unfortunately. She said that she was going to bake some Journey Bread so that Verranda wouldn't have to keep trapping something for dinner."
"A loaf of that could keep us going for a few months! You only need to eat a crumb to fill up..." I said. "How long does she think we're going to be stuck here?"
He shrugged. "Who knows? But I get to keep Solace when we do get to Menzo-"
"-Who's Solace?"
Alex shook his head. "The horse! Who else could it be? Everyone else I knew probably died back there when all nine hells broke loose..."
Ugh... Another reminder, I thought.
"Could you not bring that up?"
He shook his head. "Quit trying to pretend it didn't happen! That's not going to get you anywhere! You can try to get peace by listening to the birds, but -we - are - at - war!" He shook a finger at me. "I want to- I want to get even with them... They killed my sister, my mentor, and everyone I ever cared about... They- They... Murasa himself can beg for mercy when I'm through with him... I-!"
"-You yourself said that lying to yourself isn't going to get you anywhere! So calm down!" I didn't remember ever being so mad at someone else for being mad. We stood there for a few minutes staring at each other. It felt like a few hours.
Alexander spoke up first.
"... Sorry. The attack hit me just as hard as anyone else-"
We heard something in the forest, a russling sound, and we turned to look. It sounded like a person, so we went to investigate.
"... So that's how you guys roll..." Verranda said as soon as we got close enough to talk. I stopped, puzzled for a moment, but my eyes widened with embarrassment once I got what she suggested.
"N-No! Why would you-?"
"-Relax Lez, I was just messing with you!" She gave me one of her sly smirks. "You should've seen your face."
Alexander cleared his throat, and took a step forward. "...And you're supposed to be back at the camp. What are you doing out here?"
Verranda turned to look at him, mildly annoyed. She pulled a dead rabbit out of her makeshift belt pack.
"Just getting your dinner, that's all. So if you plan on eating, maybe you could be more polite to the only hunter here..."
"Are you saying that we couldn't survive without your rabbits? They didn't just teach me how to swing a sword at the Squire Academy, sweetie-!"
"-Don't call me 'Sweetie'!!!" Verranda immediately snapped back. She turned away from us, her face filled with hatred... But I realized it wasn't directed at Alexander, which was odd. I made a mental note to ask her about it later.
"...Look, I'm sorry. There's enough tension to go around-" Alex glanced at me as he continued. "-Let's not add to it."
Verranda sighed. "Fine."
There was a pause, and something occured to me, "... This isn't the side of the side of the forest you put your traps in... Why don't you tell Alexander and I why you're really here?"
Recovering from her anger, she let a smile poke through. "Clever, Lez. I really came to get you guys to check this cave I found. Alex has the armor, and you have the spear... Shouldn't be anything you two can't handle."
Alexander spoke up, "Wait you're afraid to go in a cave?"
Verranda laughed, "Sure! I'm afraid of these caves." She smirked.
Alex and I looked at each other. Something was up. We both knew it. We started following her, but I held my short spear tight, just in case.
~ ~
The Black Priest was in the throne room of Castle Exodus, looking out the colossal window overlooking his lake. The lake's depths were filled with darkness, an aura of fear surrounding it. Murasa knew better to stare at the lake for too long, he stepped away from the window.
It had been a long twenty years, he thought as he went to prepare himself for the evening. Perhaps tonight was going to be the most important night in all those years. Without having to deal with spies, he could continue without interruptions.
Murasa walked across the room to a table, his equipment already laid out by his servants. He browsed through the array of weapons and accessories, weighing his choices carefully.
Physical weaponry was almost useless to him. But he perferred if he wasn't completely helpless if he ran into an anti-magic field, so Murasa picked a simple mace for insurance. His Vorpal rapier would be more effective, but it wouldn't be very concealable.
The dark lord searched through the selections of power rings set before him. Most were ornate with gems set in them, some were simple metal bands. All of them were imbedded with magical properties. Murasa decided on a Ring of Spell Recovery and a Ring of True Seeing.
Murasa just had a few more accessories to pick up before he was ready. His Shadowfell Gloves. Boots of Levitation. Some Potions of Healing. And finally, his iconic Necromancer Void Cape. Few knew the secrets of this cape, he thought, and few would.
Just as he finished preparing himself, Murasa heard someone step out of the shadows to stand beside him.
"My lord?"
Murasa turned to look at him. He looked like a normal human, if not for the ram-like horns atop his head that curled down by his ears. His pointed teeth seemed plastered into an everlasting evil smile. He smelled faintly of brimstone and fire.
Murasa raised an eyebrow. "You wish to tell me something, Damakos?"
The devil-man's expression didn't change. "My lord, have I ever bothered you for something trivial? No, and I am grateful that my lord has taken notice..."
Murasa's face dimmed. Was he asking for more attention?
"... The troops are pulling out of Derynn, and are on their way inward-" Damakos' smile widened a hair. "-But my lord already knows that. What my lord doesn't know is that our men have the 'hold'."
While Damakos sometimes annoyed him, the Black Priest was pleased with the news he brang.
"... The lizards' army will no longer be any threat... Your search will bring about your everlasting reign." His smile widened a bit more.
"Excellent. Now begone."
Damakos stood like a statue. So, he thought he could ignore the Dark One?
"Damakos, go now before I have to kill you."
Damakos' smile was unchanged. The Tiefling slipped back into the shadows, bowing. "... May Orcus bless you in your search for his creation..."
Murasa relaxed himself.
"... Ragnarok-"
Startled, Murasa drew his mace and spun around instantly. His weapon swung millimeters past Damakos' fiendish grin that was waiting there. Unfased, Damakos stood there for a few seconds before making a slight bow and walking away.
Murasa was predictably furious. He gripped his mace with one hand in rage, and made it instantly disintergate. He marched over to the weapon table again. The servants immediately ran away and occupied themselves with other tasks as far from the table as possible.
The Black Priest took his Vorpal rapier and looked at the sheen of the blade. If Damakos tried that agian, he wouldn't miss: He'd sever the Tiefling's head.


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Discussion in 'Disney Collectors Board' started by Raulandpinboy, Aug 18, 2002.
Why signed up for the Great Pin Adventure 2 of course
Ok, what am I missing? Am I supposed to understand what appears to be a question ?
Log in or Sign up
to hide this advert.
I'm signed up & can't wait!
I'm with you Judith. I had to read that question/statement about 5 times. I still don't understand. But what do you expect from Raul?
I'm signed up by the way.
How many of you wonder what the hell Ed is trying to say here????? Ed; slow down, the test is over, relax, relax, relax.......now; try it again!!!!!
And yes, I will wave menus around while doing the speedo dance, I hope you all have activities scheduled for the children in another room far, far away!!!!!!
Sir Chef Manny,
I hope you are going to do the Conga in the Tonga as well!!!
Maybe after a few cosmos your highness!
Hows the brood? All well I hope.
Your royal Chef
but was apprehensive to say so. Thanks for confirming my confusion. Ask us again Ed
See, you just proved my theory. EVERYONE wants to do the SPEEDO dance, but you need a few cosmos to get into a boa.
Can't wait to see your dance. What color SPEEDO do you have? hope it doesn't clash with your eyes.
You are in for a big surprise when you see Ed in his SPEEDO. Peggie
Peggie, how do you know what Ed looks like in a Speedo????? Donna......
Pintrader, don't be shy to question Ed, he's an engineer; he is in his own little world but it's ok ,they know him there. He has his own hollowed out tree with all the creature comforts any engineer could ask for!!!!
I think my Speedo will be rainbow pattern, just to keep the coalition happy!!!!! I don't want J. Jackson on my case!!!!!
Judging from the question, I think Ed's speedo is already on and it's a little too tight.
Ya'll are cracking me UP!!!
Separate names with a comma.


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Former Chelsea striker Jimmy Floyd Hasselbaink has declared his interest in the vacant managerial position at Elland Road.
Hasselbaink, 42, who played for Leeds between 1997-99, is keen to start a managerial career and would like the chance to take charge of the club.
He told talkSPORT: "Leeds United is a wonderful, big club where I had two great years. If they want to talk to me, of course I would talk [to them].
"I've got experience, I know I could take the club forward and I know I can take the club back to where they need to go.
"But what do Leeds United themselves want? That's the most important question. How do they see things? At the moment there are so many things going on they need to sort out first, or maybe they have already.
"But if they want to talk to me, of course I would be interested. Who doesn't want to manage Leeds United? It's one of the 10 biggest jobs in the country."
Hasselbaink, who has coached in England with Nottingham Forest and managed Royal Antwerp in Belgium last season, was a key figure during his time at Elland Road. He scored 42 goals in 87 games for the club before being sold to Atletico Madrid, where he spent a season before moving to Chelsea in 2000 for £15million.
Robbie Fowler is also believed to have applied for the role following the sacking of Dave Hockaday.Reuse content


text:
How to dispose of unwanted holy books (tip: don't burn them).
Scrabble for Cheaters Tournament is this Saturday. The more you cheat, the more you'll raise for 826NYC, helping students develop writing skills.
Dear Querist: The Awl takes a look at the first English advice columns, from 1691.
The Situation: "On the Road" has been painstakingly reconstructed, sentence by sentence, for bros.
Shakespeare's fart jokes.
New Yorkers were always addicted to takeout: a historic look at the city's apartment culture.
Insights from Emma Straub on finding love in bookstores.
Famous last lines of literature. What are your personal favorites?


text:
|Publication number
|Publication type
|Application number
|Publication date
|May 18, 2004
|Filing date
|Dec 7, 1999
|Priority date
|Dec 7, 1999
|Fee status
|Publication number
|George Michalopoulos, William C. Bowen
|Original Assignee
|University Of Pittsburgh Of The Commonwealth System Of Higher Education
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (14), Non-Patent Citations (25), Referenced by (6), Classifications (27), Legal Events (7)
|External Links: USPTO, USPTO Assignment, Espacenet
The present invention relates to a novel tissue culture system that provides for the long term culture of proliferating hepatocytes that retain hepatic function. Disclosed are methods and compositions for ex vivo culturing of hepatocytes and nonparenchymal cells on a matrix coated with a molecule that promotes cell adhesion, proliferation or survival, in the presence of growth factors, resulting in a long-term culture of proliferating hepatocytes that retain hepatic function. The co-culturing method results in the formation of matrix/hepatic cell clusters that may be mixed with a second structured or scaffold matrix that provides a three-dimensional structural support to form structures analogous to liver tissue counterparts. The hepatic cell culture system can be used to form bio-artificial livers through which a subjects blood is perfused. Alternatively, the novel hepatic cell culture system may be implanted into the body of a recipient host having a hepatic disorder. Such hepatic disorders, include, for example, cirrhosis of the liver, induced hepatitis, chronic hepatitis, primary sclerosing cholangitis and alpha, antitrypsin deficiency.
The present invention is based on the discovery that mixed cultures of proliferating hepatocytes and nonparenchymal cells, grown on a collagen-coated matrix in medium containing hepatocyte growth factor (HGF) and epidermal growth factor (EGF), maintain their capacity to proliferate while retaining hepatic functions.
One of the major functions of the liver is to break down harmful substances absorbed from the intestine or manufactured elsewhere in the body, followed by their excretion as harmless by-products into the bile or blood. Abnormalities of liver function caused by insult to and/or death or malfunction of the cells in the liver can lead to a variety of different hepatic disorders including cirrhosis of the liver or hepatitis. Treatment of such disorders may include whole liver transplants, although this treatment is limited by organ availability, surgical complications, and immunologically-mediated graft rejection normally associated with liver transplantation.
While hepatocyte transplantation has been considered as an alternative to whole-organ transplantation, major technical barriers such as the inability to transfer donor hepatocytes into the liver of a recipient, in numbers to provide a beneficial result, have limited the usefulness of this approach. One of the major difficulties in constructing artificial liver tissue is that, to function effectively, the artificial liver tissue requires functionally active, differentiated hepatocytes present at high densities. Future success with artificial liver tissue will depend on the development of systems in which hepatocytes attached to matrices and packed at high density can retain long term their full functional capacity.
To generate artificial liver tissue, it will be necessary to provide in vitro cultures of hepatocytes. Unfortunately, one of the problems associated with the culturing of hepatocytes is that gene expression deteriorates rapidly as the hepatocytes proliferate. Likewise, long-term cultures of hepatocytes having stable gene expression can only be maintained in the absence of cell proliferation. Thus, one of the long-standing goals of culturing hepatocytes is the establishment of proliferating cultures with long-term gene expression.
A number of culture techniques have been developed that permit primary hepatocyte cultures to grow and/or express complex patterns of hepatocyte differentiation (Mitaka, et al., 1995, Biochem Biophys Res Commun 214: 310-317; Cable, 1997, Hepatology 26: 1444-1445; Block, et al., 1996, J. Cell Biol. 132: 1133-1149). Conditions have also been established that allow mature hepatocytes to enter into clonal expansion in cell culture (Block, et al., 1996, J. Cell Biol. 132: 1133-1149). For example, hepatocytes cultured in chemically defined hepatocyte growth medium (HGM) enter into DNA synthesis in response to polypeptide mitogens, notably epidermal growth factor (EGF), transforming growth factor-α (TGF-α), and hepatocyte growth factor (HGF). These mitogens induce multiple rounds of DNA synthesis and expansion of the cell population. The proliferating cells, however, lose most markers of hepatocyte differentiation while they retain expression of hepatocyte associated transcription factors HNF1, HNF4, and HNF3. In addition, proliferation of adult hepatocytes has been observed in serum-free medium supplemented with nicotinarnide and epidermal growth factor (EGF) (Mitaka, T., et al., 1991, Hepatology 12: 21-30; Mitaka, T., et al., 1992, Hepatology 10:440-447; Mitaka, T., et al., 1993, J. Cell Physiol, 147: 461-468; Mitaka, T., et al., Cancer Res, 1993, 53: 3145-3148; Block, G. D., et al., 1996, J. Cell Biol. 132:1133-1149; Tateno, C., et al., 1996, Am J. Pathol 148: 383-392).
Previous studies have indicated that a fundamental parameter that best determines hepatocyte gene expression in culture is the surrounding matrix. Hepatocytes embedded in complex matrices, such as Matrigel or type I collagen gels, maintain stable phenotypic expression, however, at the expense of cell proliferation. Recently, Mitaka, T. et al. (1999, Hepatology 29: 111-125) showed that small hepatocytes could differentiate to mature hepatocytes that interact with hepatic nonparenchymal cells and extracellular matrix. The mature hepatocytes reconstructed three-dimensional structures, expressed proteins known to be expressed in highly differentiated hepatocytes and the cells survived for more than 3 months, while maintaining hepatic differentiated functions. In addition, Landry et al. (1985, J. Cell Biol. 101:914-923) demonstrated the reconstruction of a three-dimensional cyto-architecture consisting of differentiated hepatocytes, bile duct-like cells and deposited extracellular matrix by the use of spheroidal aggregate culture of hepatic cells isolated from newborn rats. Three-dimensional cell culture systems have also been disordered in which hepatocytes are grown on a pre-established stromal tissue (U.S. Pat. No. 5,624,840). Attempts have also been made to grow a three-dimensional hepatic organoid using a co-culture of hepatocytes and fibroblasts (Senoo, et al., 1989, Cell Biol. Internat. Reports 13:197-206; Takezawa, et al., 1992, J Cell Sci 101:495-501).
A number of devices which perform the function of the liver and involve blood perfusion have been described (Hagger et al., 1983, ASAIO J. 6:26-35; U.S. Pat. No. 5,043,260; U.S. Pat. No, 5,270,192: Demetriou et al., 1986, Ann. Surg 9:259-271). However, a number of problems are associated with the use of such devices for treatment of patients suffering from hepatic failure or dysfunction. Perhaps, the most significant problem is the inability to culture hepatocytes that retain hepatic function for prolonged periods of time, although, attempts have been made to circumvent this problem through the use of transformed hepatocytes that are capable of proliferating indefinitely (U.S. Pat. No. 4,853,324).
Development of a stable support system that would maintain hepatic functions and be useful in stabilizing patients in partial or complete hepatic failure has been a long-term scientific goal in the field of hepatology. Similar devices have revolutionized the treatment of patients with kidney failure and have allowed long-term stabilization of a large population of patients. Currently the use of such devices in treatment of liver failure is quite limited and existing devices are based on rapidly assembled hepatocyte support systems which partially sustain the patient over a very limited period of time, i.e, 24 to 48 hours with declining function over more prolonged term use.
The present invention relates to a novel tissue culture system that provides for long term culture of proliferating hepatocytes that retain their capacity to express hepatic function. The invention generally relates to compositions and methods for generating long term cultures of hepatocytes that can be used to produce three-dimensional hepatic cell culture systems. Such hepatic cell culture systems can be used to form bio-artificial livers that function as perfusion devices. Alternatively, the three-dimensional hepatic cell cultures may be implanted into a subject having a liver disorder.
The method of the present invention comprises the co-culturing of hepatocytes and nonparenchymal cells in the presence of growth factors and a matrix material coated with at least one biologically active molecule that promotes cell adhesion, proliferation or survival. The co-culturing method results in the formation of matrix/hepatic cell clusters containing a mixture of replicating hepatocytes and nonparenchymal cells. The method of the present invention may further comprise the mixing of the matrix/hepatic cell clusters in combination with a second structured, or scaffold matrix, that provides a three-dimensional structural support to form structures analogous to liver tissue counterparts.
Compositions of the present invention include populations of matrix/hepatic cell clusters comprising co-cultures of hepatocytes and nonparenchymal cells bound to a matrix coated with at least one biologically active molecule that promotes cell adhesion, proliferation or survival. Further, the invention provides a three-dimensional hepatic cell matrix system comprising a three-dimensional support matrix containing a population of matrix/hepatic cell clusters comprising hepatocytes and nonparenchymal cells bound to a matrix coated with at least one biologically active molecule that promotes cell adhesion, proliferation or survival.
The compositions of the present invention may be used to form bio-artificial livers through which a host's blood is perfused. Alternatively, the three-dimensional hepatic cell matrix system may be transplanted to a recipient host for providing hepatic function in subjects with liver disorders. The three-dimensional matrix system is administered in an effective amount to provide restoration of liver function, thereby alleviating the symptoms associated with liver disorders. The present invention, by enabling methods for generating long-term cultures of hepatocytes, provides a safer alternative to whole liver transplantation in subjects having liver disorders including, but not limited to, cirrhosis of the liver, alcohol induced hepatitis, chronic hepatitis, primary sclerosing cholangitis and alpha,-antitrypsin deficiency.
FIGS. 1A-B. Thin sections of cells on beads in roller bottle cultures at day 15 after isolation, stained with toluidine blue.
FIG. 1A. The bead is seen as a hollow space in the center of the cell cluster. Gray material around the bead represents dense type-1 collagen deposition. The collagen surrounds and embeds connective-tissue derived nonparenchymal cells. Cells with hepatocyte morphology surround the connective tissue core.
FIG. 1B. The epithelial cells with hepatocyte morphology form an eccentric growth over a foundation of connective tissue cells. Note the formation of multiple microvilli over the hepatocytes present on the surface.
FIG. 2. Matrix deposition in Stage 1 roller bottle cultures. Panels A, B, and C show depositions of collagen types I, III, and IV, respectively. Collagen types I and III are deposited as broad bands surrounding the beads. Collagen type IV often formed basement membrane structures surrounding hepatocytes arranged in acinar or ductal configurations. Matrix is stained red whereas nuclei of the adjacent cells are stained blue. Visualization was by immunofluorescence microscopy.
FIGS. 3A-C. Electron microscopy of cultures at Stage 1 (Roller bottle).
FIG. 3A. Low magnification view of hepatocytes growing on beads, before addition of Matrigel. Hepatocytes form a continuous multilayer or monolayer culture around the beads and display circuitous, interdigitated cell-cell contacts within the abluminal membrane. Canalicular structures (CC) and tight junctions (TJ) are seen. A 1-micron thick layer of fibrillar collagen (Col) is evident between the hepatocytes' abluminal membranes and the polystyrene bead. A nonparenchymal cell (NPC) is also seen within the fibrillar collagen layer. Bar=1 mmol/L.
FIG. 3B. Another view of the cytoplasmic features of hepatocytes at stage 1 (Magnification, 4,000�). Sinusoidal endothelial cells (SEC) are forming a layer of fenestrated endothelium. Fibrillar collagen (Col) and multiple microvilli are seen under the endothelial layer, with a morphology similar to that seen in the space of Disse.
Glycogen (Gly) and lamellae of rough endoplasmic reticulum (RER) are seen in the cytoplasm of the adjacent hepatocytes.
FIG. 3C. Higher magnification of B (10,000�) showing the fenestrae of the endothelial layer. Collagen fibrils are seen in the interrupted cytoplasmic continuity of the endothelial cell at the site of the formation of the fenestra.
FIGS. 4A-C. Stains for macrophages, endothelial cells, and desmin-positive cells in Stage 1 roller bottle cultures. Visualization by differential interference microscopy. Positive immunohistochemistry is shown as red (complete arrows) whereas nuclei of cells are stained blue (truncated arrows).
FIG. 4A. Macrophages staining positive for ED-1 antigen. Note the "foamy" cytoplasm characteristic of macrophages in some of the cells.
FIG. 4B. Desmin-positive cells.
FIG. 4C. Structures of endothelial cells staining positive for 1CAM1 antigen. One of the endothelial cells contains a nucleus at the field of the image (complete arrow).
FIGS. 5A-B. Migration of cell populations from bead clusters after placement in Matrigel (Collaborative Biomedical, Mass.). Phase contrast microscopy.
FIG. 5A. Nonparenchymal cells (NP) migrate first and spread by attaching to the substratum. Occasional buddings of epithelial cells are seen at a higher focus plane (Hep). Some (arrow) appear to contain a duct. Culture at 1 week in Matrigel. Magnification, 200�.
FIG. 5B. Multiple buddings of epithelial cells migrate out of the bead clusters at different planes and in all directions. Culture at 20 days in Matrigel. Magnification, 200�.
FIG. 6. Histology of the epithelial cell buddings in Matrigel at Stage 2 cultures at day 20 in Matrigel. Epithelial cells with hepatocyte morphology (see FIG. 8) are surrounding the central bead core and are arranged in sheets and ducts. Connective tissue deposition is also present underlying the epithelial cell structures. Hematoxylin eosin stain. Magnification, 200�.
FIG. 7A. Low power electron micrograph of an acinar structure formed from the bead cluster. Evident are the duct-like canalicular structures (C) in the center of the acinar structure. Cells contain extensive RER and numerous mitochondria. A thick, but less electron dense layer of extracellular matrix than that observed for the pre-Matrigel bead is seen between the hepatocytes and the bead, with several fibroblastic (F) type cells residing in the matrix. Bar−2 mm.
FIG. 7B. High power micrograph of the canalicular structure seen in A. Readily obvious are three extensive tight junctional areas (TJ), desmosomes, RER, Golgi elements, and Mt, mitochondria. Bar=500 nm.
FIG. 8. Formation of plates by hepatocytes at Day 20 in Matrigel. Notice the prominent canalicular network (bright canals, arrows) along the middle of the plate.
FIG. 9. Cellular and matrix immunohistochemistry in Stage 2 cultures in Matrigel. Staining by immunoperoxidase. Panels A,B,C, and D show stains for desmin, Collagen types I, III, and IV, respectively. Desmin-positive stellate cells are interspersed in close proximity to the hepatocytes. Collagen type III shows the strongest immunohistochemical response. Collagen type IV often formed basement membrane structures surrounding hepatocytes arranged in acinar or ductal configurations (arrow).
FIG. 10A Phase contrast microscopy of monolayers developing at 2 to 3 months in Matrigel (Stage 3 cultures) in the presence of HGF and EGF. Magnification 100�.
FIG. 10B. Magnification 200�. Notice the extensive canalicular network (bright lines ramifying with short branches along the hepatocyte plates), the pseudo-sinusoidal spaces (S), and the duct-like structures (D).
FIG. 11A. A low power (2,000�) electron micrograph of hepatocytes in Stage 3 cultures. Notice the longitudinal section of the extensive canalicular network (with microvilli and desmosomes) surrounding the individual hepatocytes.
FIG. 11B. Higher power view (10,000�) showing detailed cytoplasmic features. Rough endoplasmic reticulum, mitochondria, and Golgi network elements are seen in the individual hepatocytes.
FIG. 12. Expression of several genes in hepatocytes immediately after isolation (Time zero), cells in roller bottle at day 13, cells in roller bottle at day 25, cells in Matrigel (Collaborative Research) cultures at day 25 (12 days after placement in Matrigel at Day 13), and nonparenchymal hepatic cell fraction (5% nonparenchymal hepatocyte contamination) immediately after isolation. Expression of GAPDH is used as a normalizing parameter.
FIGS. 13A-C. Induction of the cytochrome P450 species CYP3A (FIG. 13A), CYP1A (FIG. 13B) and CYP2B1/2 (FIG. 13C) by their characteristic inducers in day 35 cultures. The increase in actual is demonstrated by western immunoblot. C stands for control. Dex (dexamethasone); 3MC (3′ Methylcholanthrene); PB (Phenobarbital) were the inducers used correspondingly.
FIG. 14. Enzymatic Activities. The activities of testosterone 6β-hydroxylase (CYP3A dependent) and ethoxyresorufin O-deethylase (CYP1A dependent) were also measured in the same cultures. As demonstrated, more than 20-fold induction was seen in both cases by the characteristic inducers.
The present invention relates to a novel tissue culture system that provides for long term culture of hepatocytes that retain their capacity to proliferate and express hepatic function. The invention provides compositions and methods for generating long term cultures of hepatocytes that can be used as bio-artificial livers for perfusion purposes. Alternatively, the hepatic cell culture systems may be implanted into a subject having a hepatic disorder to restore or supplement liver function.
The method of the present invention comprises the co-culturing of hepatocytes and nonparenchymal cells, in the presence of growth factors and a matrix material coated with at least one biologically active capable of a molecule promoting cell adhesion, proliferation or survival, thereby, resulting in the formation of matrix/hepatic cell clusters. The method of the present invention may further comprise the mixing of the matrix/hepatic cell clusters with a second matrix material that provides a three-dimensional structural support to form structures analogous to liver tissue found in vivo.
The compositions of the present invention include matrix/ hepatic cell cultures comprising hepatocytes that retain their capacity to proliferate while expressing hepatic function. Further, the invention provides a three-dimensional hepatic cell culture system comprising hepatic cells that retain their capacity to proliferate and express hepatic function growing in a three-dimensional structure.
The hepatic cell system can be used for generating bio-artificial livers that function as perfusion devices for restoration of liver function. The three-dimensional matrix hepatic cell system can be administered to an individual for providing hepatic function in subjects with liver disorders. The matrix/hepatic cell system is administered in an effective amount necessary for restoration of liver function, thereby alleviating the symptoms associated with liver disorders.
The present invention relates to methods for generating long term cultures of proliferating hepatocytes that retain their hepatic function. The method generally comprises co-culturing or propagating hepatocytes and nonparenchymal cells on a matrix coated with a biologically active molecule that promotes cell adhesion, in vitro. The cells are cultured under conditions effective and for a time sufficient to allow formation of a culture of proliferating hepatocytes that retain hepatic function. The cells are grown in the presence of growth factors that maintain hepatic cell differentiation and the capacity to proliferate.
Hepatocytes and nonparenchymal cells may be obtained from a variety of different donor sources. In a preferred embodiment, autologous cells are obtained from the subject who is to utilize the bio-artificial liver or receive the transplanted hepatic cells to avoid immunological rejection of foreign tissue. In yet another preferred embodiment of the invention, allogenic liver tissue for use in purifying cells may be obtained from donors who are genetically related to the recipient and share the same transplantation antigens on the surface of their hepatic cells. Alternatively, if a sibling is unavailable, tissue may be derived from antigenically matched (identified through a national registry) donors.
In an embodiment of the invention, hepatic cells and nonparenchymal cells are isolated from a disaggregated liver tissue biopsy. This may be readily accomplished using techniques known to those skilled in the art. For example, the liver tissue can be disaggregated mechanically and/or treated with digestive enzymes and/or chelating agents that weaken the connections between neighboring cells, making it possible to disperse the tissue suspension of individual cells. Enzymatic dissociation can be carried out by mincing the liver tissue and treating the minced tissue with any of a number of digestive enzymes. Such enzymes include, but are not limited to, trypsin, chymotrypsin, collagenase, elastase and/or hylauronidase. A review of tissue disaggregation techniques is provided in, e.g., Freshney, Culture of Animal Cells, A Manual of Basic Technique, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch. 9, pp.107-126. In addition to primary cell cultures, established hepatic cell lines may also be utilized in the methods and compositions of the invention.
The present methods and compositions can also employ hepatic cells genetically engineered to enable them to produce a wide range of functionally active biologically active proteins, including but not limited to growth factors, cytokines, hormones, inhibitors of cytokines, peptide growth and differentiation factors. Additionally, the cells may be genetically engineered to increase their proliferative capacity, i.e, the cells may be immortalized. Methods which are well known to those skilled in the art can be used to construct expression vectors containing a nucleic acid encoding the protein coding region of interest operatively linked to appropriate transcriptional/translational control signals. See, for example, the techniques described in Sambrook, et al., 1992, Molecular Cloning, A Laboratory Manuel, Cold Spring Harbor Laboratory, N.Y., and Ausebel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates & Wiley Interscience, N.Y., incorporated herein by reference.
Once isolated, the hepatic and nonparenchymal cells can be grown in any culture medium known to those skilled in the art to support the growth and proliferation of such cells. For example, the mixed cultures of cells can be grown in chemically defined hepatocyte growth medium (HGM) supplemented with specific growth factors and regulatory factors. Such factors can be added to the culture media to enhance, alter or modulate proliferation and/or differentiation of the cultured hepatocytes and nonparenchymal cells. In a preferred embodiment of the invention, the culture media may be supplemented with growth factors such as hepatocyte growth factor (HGF) and/or epidermal growth factor (EGF), or functional homologs thereof, to impart phenotypic stability in terms of differentiated hepatocyte gene expression and the ability to proliferate.
In addition, the co-cultures of cells are propagated in the presence of a natural or synthetic matrix that provides support for hepatic cell growth during in vitro culturing. The type of matrix that may be used in the practice of the invention is virtually limitlessness. The matrix will have all the features commonly associated with being "biocompatible", in that it is in a form that does not produce an adverse, or allergic reaction when administered to the recipient host. In a preferred embodiment of the invention, the matrix is in the form of a bead to which the cultured cells may adhere. The beads may be composed of variety of different substances including, but not limited to, synthetic materials or naturally derived materials. The type of matrix material to be used will depend on the desired use of the hepatocyte cultures. For example, when the matrices are to be transplanted into a subject it is preferred that a biodegradable matrix material be used. For purposes of forming bio-artificial livers, the matrix may be composed of any suitable material to which the hepatocytes and nonparenchymal cells will adhere and proliferate.
Further, to improve hepatic cell adhesion, proliferation or survival, the matrix is coated on its external surface with factors known in the art to promote cell adhesion, growth or survival. Such factors include cell adhesion molecules, extra-cellular matrix molecules and/or growth factors for hepatocytes and/or nonparenchymal cells. Matrices may also be designed to allow for sustained release of growth factors over prolonged periods of time. Thus, appropriate matrices will ideally provide factors known to promote hepatic cell adhesion, growth or survival, and also act as a support on which the cultured cells differentiate and proliferate. In a preferred embodiment of the invention, the hepatic cell cultures are propagated in media containing matrices coated with collagen type I protein for promotion of cell adhesion and proliferation of bound hepatocytes.
The method of the present invention involves the co-culturing of hepatic and nonparenchymal cells in the presence of the selected matrix material. Although the cells may be propagated under static conditions, it is preferred that the cells arc propagated under mixing or stirring conditions wherein a cell suspension is combined with matrix, and mixed or stirred, to enhance the number and frequency of cell contacts with the matrix to maximize cell adhesion to the matrix, but not disrupt adherence to cells. Such conditions may be generated in variety of different ways including, for example, the use of roller bottles to provide continuous stirring or mixing of the culture. Preferably, the stirring is continued throughout the culturing of the hepatic and nonparenchymal cells.
The conditions of long-term matrix-cell culturing will preferably be maximized to enhance hepatocyte proliferation while maintaining hepatic function. Although certain variations in cell number, seeding techniques, culture media, incubation temperatures and incubation times, may be utilized, such variations would be routine to those skilled in the art and are encompassed by the present invention.
The present invention further relates to the use of the matrix/hepatic/nonparenchymal cell clusters, produced as described in Section 5.1, for generation of three-dimensional hepatic cell culture systems to form structures analogous to liver tissue counterparts. The method of the invention comprises growing hepatic and nonparenchymal cells on a three-dimensional matrix in vitro under conditions effective and for a period of time sufficient to allow proliferation of the cells to form a three-dimensional structure.
The three-dimensional matrices to be used are structural matrices that provide a scaffold for the cells, to guide the process of tissue formation. Cells cultured on a three-dimensional matrix will grow in multiple layers to develop organotypic structures occurring in three dimensions such as ducts, plates, and spaces between plates that resemble sinusoidal areas, thereby forming new liver tissue. Thus, in preferred aspects, the present invention provides a three-dimensional, multi-layer cell and tissue culture system. The resulting liver tissue culture system survives for prolonged periods of time and performs liver-specific functions for use as a perfusion device or following transplantation into the recipient host.
A wide variety of structural matrices may be used in the context of the present invention for preparation of a three-dimensional hepatic cell culture system. In preferred embodiments, the matrices are bio-compatible matrices that provide a scaffold for the cells to guide the development of tissue. Preferred matrices are generally those that define a space for subsequent tissue development. Such matrices include hydrogels, biomatrix gels, or porous materials such as fiber based or sponge like matrices. The culture system described herein provides for the proliferation of cells to form structures analogous to liver tissue counterparts in vivo.
In certain embodiments, synthetic matrices, such as synthetic polymer matrices, may be used. Such matrices include, but are not limited to, nylon, dacron, polystyrene and homopolymers or heterpolymers such as polylactic acid (PLA) polymers, polyglycolic acid (PGA) polymers and polylactic acid-polyglycolic acid (PLGA) copolymer matrices. In other embodiments, matrices for use in the invention may be naturally-derived matrices extracted from or resembling extracellular matrix materials such as a collagen matrix, such as type I collagen. Other naturally derived matrix materials include laminin-rich gels, alginate, agarose and other polysaccharides, gelatin and hyaluronic acid derivatives. Certain matrix materials may not support efficient cellular attachment and, in such instances, it may be advantageous to coat the matrix with molecules that promote cell adhesion, such as extracellular matrix proteins or, specifically, collagen type I.
To generate the three-dimensional hepatic cell cultures, matrix/hepatic/nonparenchymal cell clusters generated as described above in Section 5.1 are isolated from cell culture suspensions. For example, the cell clusters may be isolated by low speed gravity sedimentation. The matrix/hepatic/nonparenchymal cell clusters are then exposed to a second structural matrix material in the presence of an appropriate culture media, thereby providing an environment for three-dimensional hepatic cell growth. Many commercially available culture media, supplemented in some instances with growth factors and the like, may be suitable for use. In addition, the culture media may be replenished periodically to provide a fresh supply of nutrients. The three-dimensional hepatic cell culture system is cultured for a sufficiently long period of time to allow the hepatic cells to replicate to form a three-dimensional cell or tissue structure.
Prior to use of three-dimensional hepatic cell cultures, the cultures may be contacted with a number of different growth factors that can regulate tissue regeneration by affecting cell proliferation, and gene expression. Such growth factors include those capable of stimulating the proliferation and/or differentiation of hepatic progenitor cells. For example, epidermal growth factor (EGF), transforming growth factor α (TGF-α) or hepatocyte growth factor (HGF) may be utilized. The hepatic cells may be stimulated in vitro prior to transplantation into the recipient subject, or alternatively, by injecting the recipient with growth factors following transplantation.
The hepatic cell cultures of the invention can be used as bio-artificial livers for use by subjects having liver disorders that result in hepatic failure or insufficiency. The use of such bio-artificial livers involves the perfusion of the subject's blood through the bio-artificial liver. In the blood perfusion protocol, the subject's blood is withdrawn and passes into contact with the hepatocyte cell cultures. During such passage, molecules dissolved in the patient's blood, such as bilirubin, are taken up and metabolized by the hepatocyte cultures. In addition, the cultured hepatocytes provide factors normally supplied by liver tissue.
To form the bio-artificial liver the three-dimensional hepatocyte cell cultures of the invention are grown within a containment vessel containing an input and output outlet for passage of the subjects blood through the containment vessel. The bio-artificial liver further includes a blood input line which is operatively coupled to a conventional peristaltic pump. A blood output line is also included. Input and output lines are connected to appropriate arterial-venous fistulas which are implanted into, for example, the forearm of a subject. In addition, the containment vessel may contain input and output outlets for circulation of appropriate growth medium to the hepatocytes for continuous cell culture within the containment vessel.
In an embodiment of the invention, semipermeable membranes may be included in the bio-artificial livers to prevent direct contact of the subject's blood with the three-dimensional hepatocyte cultures. In such instances, the molecules dissolved in the subject's blood will diffuse through the semipermeable membrane and are taken up and metabolized by the hepatocycte cultures.
The use of the cultured hepatocyte systems of the invention to form bio-artificial livers provides a method which may be utilized to provide liver function to subjects suffering from hepatic failure or insufficiency.
The three-dimensional hepatic cell cultures can also be administered or transplanted to the recipient in an effective amount to achieve restoration of liver function, thereby alleviating the symptoms associated with liver disorders. When the hepatic cell cultures are to be administered to a recipient, it is desirable to form the hepatocyte cultures with hepatocytes and nonparenchymal cells derived from the recipient so as to avoid tissue rejection.
The number of cells needed to achieve the purposes of the present invention will vary depending on the degree of liver damage and the size, age and weight of the host. For example, the cells are administered in an amount effective to restore liver function. Determination of effective amounts is well within the capability of those skilled in the art. The effective dose may be determined by using a variety of different assays designed to detect restoration of liver function. The progress of the transplant recipient can be determined using assays that include blood tests known as liver function tests. Such liver function tests include assays for alkaline phosphatase, alanine transaminase, aspartate transaminase and bilirubin. In addition, recipients can be examined for presence or disappearance of features normally associated with liver disease such as, for example, jaundice, anemia, leukopenia, thrombocytopenia, increased heart rate, and high levels of insulin. Further, imaging tests such as ultrasound, computer assisted tomography (CAT) and magnetic resonance (MR) may be used to assay for liver function.
The three-dimensional hepatic cell system can be administered by conventional techniques such as injection of cells into the recipient host liver, injection into the portal vein, or surgical transplantation of cells into the recipient host liver. In some instances it may be necessary to administer the hepatic cell composition more than once to restore liver function. In addition, growth factors, such as G-CSF, or hormones, may be administered to the recipient prior to and following transplantation for the purpose of priming the recipients liver and blood to accept the transplanted cells and/or to generate an environment supportive of hepatic cell proliferation.
The purpose of the present example is the demonstration that mixed cultures of hepatocytes and nonparenchymal cells grown in chemically defined hepatocyte growth medium (HGM) containing hepatocyte growth factor and epidermal growth factor on collagen-coated polystyrene beads retain their hepatic functions while maintaining their capacity to proliferate.
Male Fischer 344 rats from Charles River were used for the studies described.
EGF was obtained from Collaborative Biomedical (Waltham, Mass.).
Collagenase for hepatocyte isolation was obtained from Boehringer Mannheim (Mannheim, Germany). Vitrogen (Celtrix Labs., Palo Alto, Calif.) was used for the construction of the collagen gels. General reagents were obtained from Sigma (St. Louis, Mo.). EGF and Matrigel (Collaborative Research) were purchased from Collaborative Biomedical (Waltham,. Mass.). HGF used for these studies was the Δ5 variant and was kindly donated by Snow Brand. (Toshigi, Japan). Polystyrene beads coated with type I collagen were purchased from SoloHill Inc. (Ann Arbor, Mich.). Antibody sources: Mouse anti-rat ICAM (CD54) Pharmingen (San Diego, Calif.) (1:500); rabbit anti-rat collagen I, Chemicon (Temecula, Calif.) (1:100); rabbit anti-rat collagen III, Chemicon (1:100); Mouse anti-desmin, Dako (Carpenteria, Calif.) (1:100); Mouse anti-rat monocyte/macrophage (ED-1) Serotec (Raleigh, N.C.) (1:500); Rabbit anti-rat Collagen IV, gift from Dr. A. Martinez-Hernandez (1:100).
Rat hepatocytes were isolated by an adaptation of Seglen's calcium two-step collagenase perfusion technique (Seglen, P. O., 1976, Methods in Cell Biol. 13:29-83) as previously described (Kost, D P et al., 1991, J. Cell Physiol. 147:274-289). Typically, a 3% contamination with nonparenchymal cells is seen in this isolate.
The nonparenchymal cell fraction was defined as the cell pellet isolated from the supernatant of the first low-gravity centrifugation used to prepare hepatocytes. This fraction primarily contains cells of Ito, bile duct cells, and endothelial cells. Small hepatocytes are also present in this fraction, typically comprising 5% of the cells.
Freshly isolated hepatocytes were added to roller bottles (850 cm2 surface) obtained from Falcon (Franklin Lakes, N.J.). Each bottle contained 18.7�106 polystyrene beads and 210�106 freshly isolated hepatocytes in 250 mL of HGM medium supplemented with HGF (20 ng/mL) and EGF (10 ng/mL). The bottles were rotated at a rate of 2.5 rotations per minute and kept in an incubator maintained at 37� C., saturated humidity, and 5% CO2. The viability of the cultures was assessed by periodic sampling. The samples were directly observed under a phase contrast microscope as well as stained with methyl tetrazolium to assess viability.
The bead clusters containing cells were isolated from suspensions obtained from the roller bottle cultures. Enrichment for clusters was obtained by allowing for 2 minutes of unit gravity sedimentation. The bead and cell clusters were mixed with Matrigel (Collaborative Research). Bead clusters with attached cells were allowed to settle whereas beads without cells stayed mostly in suspension. The supernatant was aspirated leaving the, clusters in the bottom of the tube. The process was repeated three times. Clusters suspended in medium were mixed with Matrigel at a volume ratio of 1:4 (medium plus beads: Matrigel). Approximately 50 to 100 bead clusters were randomly embedded in Matrigel.
HGM was prepared as previously described (Block, G. D. et al., 1996, J. Cell Biology 132:1133-1149). DMEM medium powder, HEPES, glutamine, and antibiotics were purchased from GIBCO/BRL (Grand Island, N.Y.). ITS mixture (Insulin, Transferrin, Selenium) was purchased from Boehringer Mannheim. All other additives were cell-culture grade (Sigma). Unless otherwise indicated for specific experiments, the basal HGM consisted of DMEM supplemented with purified bovine albumin (2.0 g/L), glucose (2.0 g/L), galactose (2.0 g/L), ornithine (0.1 g/L), proline (0.030 g/L), nicotinamide (0.305 g/L), ZnCl2 (0.544 mg/L), ZnSO4; 7H2O (0.750 mg/L), CuSO4:5H2O (0.20 mg/L), MnSO4 (0.025 mg/L), glutamine (5.0 mmol/L), and dexamethasone (10−7 mol/L). Penicillin and streptomycin were added to the basal HGM at 100 Mg:/L and 100 μg/L, respectively. The mixed basal HGM was sterilized by filtration through a 0.22-μm low-protein—binding filter system, stored at 4� C., and used within 4 weeks. ITS 1.0 g/L, (right hip-insulin 5.0 mg/L, human transferrin 5.0 mg/L [30% diferric iron saturated], selenium 5.0 μg/L) was added after filtration immediately before use. The growth factors, as required, were added to HGM fresh at the specified concentrations every time the medium was changed.
Samples for transmission electron microscopy were washed once in PBS with 1 mmol/L MgCl2, 0.5 mmol/L CaCl2, then fixed overnight at 4� C. in 2.5% glutaraldehyde in PBS. Samples were washed three times with PBS then postfixed in 1% OsO4, 1% KFe(CN)6 in PBS for 1 hour at room temperature. Samples were washed three times in PBS, then dehydrated through graded series (30%-100%) of ethanol. Following three changes of 100% ethanol, samples were infiltrated with several changes of Polybed 812 resin (Polysciences, Warrington, Pa.) at room temperature, a change overnight at 4� C., then a final change, in the case of cells grown on monolayers, where Beem capsules, filled with resin, were inserted on top of areas of interest. Resin was hardened overnight at 37� C., then for 2 additional days at 65� C. While the resin was still warm, Beem capsules were pulled from the dish and analyzed to ensure that the cells did not remain on the dish. In some cases monolayers were re-embedded to obtain cross sections. Thick sections (300 μm), obtained using a Reichert (Vienna, Austria) ultramicrotome fitted with a diamond knife, were heated onto glass slides, stained with 1% Toluidine Blue, and rinsed with water. Ultra thin sections (60 nm) were collected on Formvar-coated (Fullam, Schenectady, N.Y.) grids and stained with 2% uranyl acetate in 50% methanol for 10 minutes, then 1% lead citrate for 7 minutes. Sections were analyzed and photographed on a JEOL JEM 1210 transmission electron microscope at 80 kV.
Samples from roller-bottle cultures were fixed in 2% paraformaldehyde and 0.01% glutaraldehyde in PBS for 1 hour. Liver seeds were then stabilized by dipping them in 3% gelatin in PBS, then refixing them in the above fixative for an additional 15 minutes. Samples were incubated in 2.3 mol/L sucrose in PBS at 4� C. overnight. Samples were mounted on screw stubs and snap-frozen in liquid nitrogen. Five hundred nanometer-thick frozen sections were cut on a FCS Ultracut Microtome (Reichert) fitted with a cryokit. Sections were attached to glass slides by adsorbed Cell-Tak (Collaborative Biomedical). Sections were washed in 0.5% BSA, 0.15% glycine in PBS (PBG buffer) three times to remove sucrose, then blocked with 5% goat serum in PBG buffer for 30 minutes. Sections were then stained with various antibodies in PBG buffer for 1 hour at room temperature, washed three times in PBG buffer then stained with Cy3-conjugated (goat antirabbit or antimouse) secondary antibodies (Jackson Immunolabs, Bar Harbor, Me.) for 1 hour. Sections were washed three times with PBG buffer, then once in PBS. Nuclei were stained with 0.1 mg/mL Hoechst (bisBenzimide) for 30 seconds, washed twice with PBS, then mounted on slides with use of gelvatol (23 g polyvinyl alcohol 2000, 50 mL glycerol, 0.1% sodium azide to 100 mL PBS), and viewed on an Olympus Provis epifluorescence microscope (Olympus America, Melville, N.Y.) also equipped for differential interference microscopy.
Total RNA was extracted by use of RNAzol B� (Biotecx, Houston, Tex.). RNA extraction from roller-bottle cultures was performed by washing bead-cell clusters in phosphate buffered saline and further digestion of the clusters by adding an equal volume of Trypsin-Ethylenediaminetetraacetic acid (GIBCO-BRL) to the bead-cell suspension. The mixture was shaken at 37� C. for 10 minutes. The bead-cell clusters were further washed in S+M buffer at 4� C. three times. The bead-cell pellet was mixed with three volumes of RNAzol and purified according to the manufacturer's guidelines.
RNA was extracted from Matrigel (Collaborative Research) -embedded beads by vortexing using 2.0 mL of RNAzol B� (Biotecx) per 1 mL of beads in Matrigel and purified per the manufacturer's guidelines. RNA concentration and purity were determined routine spectrophotometry. Size separation of 20 μg RNA per lane was completed on denaturing 1% agarose gels and transferred to nylon membranes (Amersham, Piscataway, N.J.) by the capillary method. After cross-linking under ultraviolet light, membranes were hybridized overnight with specific complementary DNAs (as indicated in FIG. 12) that had been labeled with [α-32P]dCTP using Amersham random primer kit. Membranes were subsequently washed under high stringency conditions and exposed to R film (photographic film) (Kodak, N.Y.) for 1 to 3 days. Quantification of the RNA hybridization bands was performed by laser densitomer.
EGF-R (rat) was obtained from Dr. Sheldon Earp, University North Carolina at Chapel Hill; acidic fibroblast growth factor receptor from American Type Culture Collection (catalog number 78222); acidic fibroblast growth factor receptor from American Type Culture Collection (catalog number 65796); urokinase plasminogen activator originated from Dr. Jay Degen, University of Cincinnati; cytochrome IIB1 from Dr. Steve Strom (University of Pittsburgh); complementary DNAs for albumin, α-fetoprotein were generated by Dr. Joe Locker (University of Pittsburgh).
Stage 1: Cultures of Hepatocytes on Beads in Roller Bottles. Collagen-coated polystyrene beads, were placed in roller bottles at a ratio of 18.7�106 beads to 210�106 freshly isolated hepatocytes. HGF and EGF were added as standard supplements in the HGM medium of the roller bottle cultures. Cells attached to the beads and, within 2 to 3 weeks, formed clusters of beads bound together with mesenchymal cells surrounded by layers of epithelial cells. The mesenchymal cells concentrate toward the center of the cluster and surround the individual beads (FIGS. 1A and 1B). They are associated with heavy deposition of type I and type III collagen immediately against the surface of the bead (FIG. 2). The collagen bundles surround the mesenchymal cells. Collagen type IV was seen as a thin rim forming a basement membrane surrounding only acinar structures of epithelial cells. The epithelial cells grow outside of the mesenchymal cells and symmetrically surround the beads or make eccentric projections. The epithelial cells have characteristics of small mature hepatocytes, as shown by electron microscopy. They contain multiple mitochondria and minimal rough endoplasmic reticulum (FIG. 3). Mature bile canaliculi containing microvilli as defined by junctional complexes were occasionally seen. Most often, they appeared as spaces surrounded by hepatocytes and containing microvilli. The junctional complexes were not as clearly defined as after placement in Matrigel (Collaborative Research). Those cells that are on the surface of the clusters have visible microvilli, whereas those toward the interior do not. The epithelial cells form multiple cell layers from the mesenchymal cell layer of the cluster to the surface. The cytoplasmic details of the epithelial cells in the clusters are shown in FIG. 3B and 3C. Multiple lamellae of rough endoplasmic reticulum and glycogen deposition is seen. Notable is the occasional information of fenestrated endothelium surrounding the hepatocytes. The proliferating cellular nuclear antigen (PCNA) labeling index of the epithelial cells exceeded 70% in all clusters. The BRdU labeling index of epithelial cells varied from 10% to 15% in different clusters. The number of nonparenchymal cells varied from one cluster to another. FIG. 4 shows desmin-positive mesenchymal cells, presumably derived from stellate cells contaminating the original hepatocyte preparation, interspersed between the epithelial cells. Approximately 15% to 20% of the cells at this stage seem to belong to this category. ICAM1-positive endothelial cells are also seen in FIG. 4, occasionally forming ICAM1-positive luminal structures. Overall, less than 2% of the cells at this stage stained positive for this antibody. Macrophages, identified as ED-1-positive cells, are seen only insporadic clusters, representing less than 0.1% of the total cell population.
Stage 2: Cultures in the First 3 Weeks After Implantation in Matrigel. Clusters of beads with the mixed cell populations were placed in Matrigel (Collaborative Research) as described in Materials and Methods. This resulted in a series of cell migrations. Mesenchymal cells with stellate shape migrated out of the beads first at about day 4 to 5 and in many instances formed a mat surrounding the beads (FIG. 5A). Protrusions with rounded contours, appearing as buds, were seen extending randomly in all directions from the bead clusters at about day 7 to 10. Some of them (approximately 30%) appeared to contain ducts. The typical appearance of these cultures is shown in FIG. 5B. Sections of these bud structures stained with hematoxylin and eosin are shown in FIG. 6. The buds consisted primarily of hepatocytes arranged in acinar structures or in sheets. Electron microscopy (FIG. 7) showed enhanced cytoplasmic differentiation of hepatocytes compared with cells in the roller bottle. Hepatocytes in the buds contained abundant lamellae of rough endoplasmic reticulum, glycogen, and canaliculi with complete junctional complexes. The latter features are not seen in the hepatocytes before implantation in Matrigel. In most cultures, several long plates, 1 to 2 hepatocytes in width and 10 to 20 hepatocytes in length (FIG. 8), were seen. These structures averaged about 20 to 30 per plate, with plates of different length extending from most clusters. The plates typically developed into areas of the substratum that were free of other cell types. There were no visible nonparenchymal cells underlying or surrounding these plates. A typically demarcated and fully developed canalicular network was seen along the entire length of the plates. Many of these single plates contained ducts at the end. IL6 (10 ng/mL) added to the cultures augmented the number of duct structures and caused formation of ducts along the plates or in the monolayer patches of hepatocytes. TGF-β1 (at 0.5 ng/mL) inhibited formation of all structures that developed from epithelial cells (buds, plates, and ducts) though migration of the nonparenchymal cells was not inhibited. The full spectrum of changes was seen in the presence of HGF plus EGF. Cultures maintained in HGF or EGF alone showed fewer and more limited changes per cluster compared with those with both growth factors. The extensive budding of the epithelial cells was associated with cell proliferation as judged by staining for PCNA. The numbers of labeled hepatocytes in the Matrigel ranged from 40% to 80% of epithelial cells per cluster, with considerable variation seen from one site to the next or among clusters. The BRdU labeling index, indicating active DNA synthesis, varied from 10% to 15% per cluster. Desmin-positive cells were seen interspersed and surrounding the hepatocytes. Type IV collagen was seen often as a thin rim surrounding acinar structures of hepatocytes. Slight staining was seen for type I and stronger staining for type III collagen (FIG. 9).
Stage 3: Long-Term Cultures in Matrigel. Long-term follow-up showed that HGF or EGF added separately was not sufficient to maintain prolonged viability of the epithelial cells. By 3 months, no epithelial cells were present in cultures maintained in HGF or EGF alone, or in control cultures without the addition of growth factors. In cultures maintained with combined HGF plus EGF, large monolayer patches of hepatocytes ranging from 2 to 10 mm in diameter were seen (FIG. 10). These structures appear at the rate of 2 to 4 patches per plate. These patches had a cyto-architecture of striking similarity to sections of the liver acinus. Single or double hepatocyte plates were seen extending in a linear or convoluted manner. Complete canalicular networks developed throughout the entire length of each of the plates. The plates were separated by spaces that, though resembling the sinusoidal spaces seen in the liver lobules, did not contain any cells. Occasional ducts were also present in random locations along the plate structures. Electron microscopy (FIG. 11) showed typical hepatocyte morphology with most features typically present in hepatocytes, including glycogen, abundant rough endoplasmic reticulum, microbodies, and bile canaliculi with mature junctional complexes.
Gene Expression Changes in Cultures at Stages 1 and 2. The expression of several genes was examined in cultures at stages 1 and 2. Monolayers at stage 3 were not available in sufficient numbers for RNA preparation. FIG. 12 compares expression of several genes in hepatocytes and nonparenchymal cells immediately after isolation from liver, cells from roller bottle cultures at day 13, cells from roller bottle cultures at day 25, and cell-bead clusters at 12 days after implantation in Matrigel (Collaborative Research) (day 25 after cell isolation). The first and last lanes show expression of the same genes respectively in hepatocytes and the nonparenchymal cell fraction, immediately after isolation from the rat liver. (several hepatocyte associated genes are expressed in this fraction as a result of contamination by small hepatocytes). Through Matrigel-enhanced expression of α-fetoprotein, cultures in the roller bottles and in Matrigel maintained high expression of albumin. EGF-receptor expression decreased in culture, whereas HGF-receptor expression was maintained in roller bottles and in Matrigel, though Matrigel caused a decrease in c-met expression, CYPB1 expression decreased gradually in the roller bottle cultures but was restored after addition of Matrigel. TGF-β1 expression, derived from the nonparenchymal cells present in the mixed cultures, was pronounced in the roller bottle cultures at stage 1 but suppressed by Matrigel in stage 2 cultures. The same was true for urokinase plasminogen activator and its receptor urokinase plasminogen activator-R Expression of transferrin and α-1 antitrypsin was also enhanced at stage 2. A separate study was conducted to evaluate induction of cytochrome P450 species in stage 1 cultures. Induction of cytochrome P450 species CYP1A, CYP3A, CYP2B1/2 was seen in response to 3′ Methyl-cholanthrene, Dexamethasone, and Phenobarbital, respectively.
FIGS. 13A-C demonstrates induction of the cytochrome P450 species CYP3A (FIG. 13A), CYP1A (FIG. 13 B) and CYP2B1/2 (FIG. 13C) by their characteristic inducers in day 35 cultures. The increase demonstrated by western immunoblot. Dexamethasone, methylcholanthrene) and phenobarbital were the inducers used correspondingly. The activities of testosterone 6β-hydroxylase (CYP3A dependent) and ethoxyresorufin O-deethylase (CYP1A dependent) were also measured in the same cultures. As demonstrated in FIG. 14, more than 20-fold induction was seen in both cases by the characteristic inducers.
The present invention is not to be limited in scope by the specific embodiments described herein which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the claims. Various publications are cited herein, the contents of which are hereby incorporated, by reference, in their entireties.
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|International Classification
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|Mar 20, 2000
Owner name: PITTSBURGH, UNIVERSITY OF, PENNSYLVANIA
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text:
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Billionaire Mavericks owner and Shark Tank host Mark Cuban has made a bold prediction about Donald Trump if he does not win the November election against Hillary Clinton. His prediction is that Trump will actually go broke and bankrupt seven years following his defeat, claiming "that's how toxic his brand now is."
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text:
Università della Svizzera italiana as an organisation
Università della Svizzera italiana (USI) was established in 1996 as an independent institution offering a public service. It is operated as a private company, with transparency and efficiency. Its young and agile organisational structure ensures smooth procedures and fast decision-making.
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text:
If you need a convenient kitchen gadget to cook your eggs with ease, the swan eggs boiler and poacher will be a perfect choise for you.
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September 26, 2013
Leona Lewis and Owain Yeoman Photo Campaign Says Bunnies are for Hugging, Not Hurting in Cosmetics Tests
"The Mentalist" star Owain Yeoman and Grammy-nominated singer Leona Lewis are showing bunnies some love this International Rabbit Day.
September 26, 2013
Martha Stewart Calls on N.J. Legislators to Help Protect Pigs from Cruelty
New Jersey native Martha Stewart has written to members of the New Jersey legislature on behalf of breeding pigs.
August 5, 2013
"Keiko: The Untold Story of the Star of Free Willy"
Celebrate the 20th anniversary of the Genesis Award-winning film "Free Willy" with the world premiere of "Keiko: The Untold Story of the Star of Free Willy."
May 24, 2013
Pets of Valor Award Finalists Announced, Online Voting Now Open
The Humane Society of the United States is pleased to announce the top five finalists for the Pets of Valor Award. The award celebrates the human-animal bond by honoring rescued or adopted dogs and cats who have exhibited an extraordinary sense of courage or resolve by heroically helping a person in need.
May 24, 2013
Se anuncian los finalistas para el Premio 'Pets of Valor', votación en línea ya está abierta
La organización de protección animal The Humane Society of the United States se complace en anunciar a los cinco finalistas para el premio 'Pets of Valor'. El premio celebra el vínculo humano-animal al honrar a perros y gatos rescatados que han mostrado un extraordinario sentido de valentía al ayudar heroicamente a una persona necesitada.
March 28, 2013
Saving the Whales in "Big Miracle"
"Big Miracle," inspired by historic whale rescue, shows the power of the human/animal bond and the power of media to help animals. It also tells a great story.
March 27, 2013
The Wyler Award
Named in memory of the late Genesis Awards founder, animal advocate and Broadway headliner Gretchen Wyler, the prestigious Wyler Award is bestowed on a celebrity or public figure who has made news on behalf of animals.
March 19, 2013
La HSUS Busca Perros o Gatos Heroicos para el Premio "Pets of Valor"
La HSUS está aceptando nominaciones para el Premio "Pets of Valor" (mascotas valientes), la cual honra a perros y gatos rescatados que han mostrado un extraordinario sentido de valentía al ayudar heroicamente a una persona necesitada.
March 14, 2013
Humane Lobby Day Kicks Off in Austin with Special Guest Kyle Chandler and Daughter Sawyer
Texans concerned about animal welfare, including actor Kyle Chandler, converged on the state capitol for Humane Lobby Day to meet with lawmakers and lobby for better animal protection laws.
March 12, 2013
The HSUS to Honor LUSH Cosmetics with Genesis Corporate Award
The HSUS will bestow the inaugural Genesis Corporate Award on the global cosmetics company LUSH for raising awareness of animal protection issues through its ongoing commitment to creating cruelty-free products and for strenuously campaigning for animal-free testing in Europe and around the world