ognizant Communication Corporation



Cell Transplantation, Vol. 10, pp. 665-671
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Co-Grafts of Fetal Ventral Mesencephalon and Fibroblasts Expressing Sonic Hedgehog: Effect on Survival and Function of Dopamine Grafts

David M. Yurek,1 Anita Fletcher-Turner,1 Jennifer Moore,1 Ling Chai,2 and Nagesh Mahanthappa2*

1Department of Surgery/Neurosurgery, University of Kentucky College of Medicine, Lexington, KY 40536-0305
2Curis, Inc., 45 Moulton Street, Cambridge, MA 02138

Fibroblasts derived from the Rat2 parental cell line were genetically modified to express the cell-associated form of Sonic hedgehog (Shh) and then co-grafted along with E14 fetal ventral mesencephalon (VM) tissue into the denervated striatum of F344 rats; fetal VM grafts alone or co-grafts using the nonexpressing Rat2 fibroblasts served as controls. Seven weeks after grafting, co-grafts of fetal VM and fibroblasts expressing Shh (Rat2/Shh) contained significantly more tyrosine hydroxylase-positive (TH+) neurons than either the fetal VM grafts or co-grafts of fetal VM plus nonexpressing fibroblasts (Rat2). Despite a significantly higher yield of grafted TH+ neurons in the fetal VM + Rat2/Shh co-grafts than in either of the other two control groups, amphetamine-induced rotational behavior scores were not significantly different between any of the three treatment groups. The number of TH+ neurons in the Rat2 (nonexpressing) co-grafts was significantly lower than the other two treatment groups. The results from this study suggest that fibroblasts expressing Shh may improve the number of co-grafted dopamine neurons, but do not improve the functional capacity of the graft in terms of improving amphetamine-induced rotational behavior.

Key words: Neural transplantation; Sonic hedgehog; Parkinson's disease; Dopamine; Rodent; Genetically modified fibroblasts; Fetal ventral mesencephalon

Address correspondence to David M. Yurek, Ph.D., Department of Surgery/Neurosurgery, University of Kentucky College of Medicine, Health Sciences Research Building, Lexington, KY 40536-0305. Tel: (859) 257-8219; Fax: (859) 323-6343; E-mail: dyure00@pop.uky.edu

*Current address: Vertex Pharmaceuticals, Inc., 130 Waverly Street, Cambridge, MA 02139.

Cell Transplantation, Vol. 10, pp. 673-680
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Subretinally Transplanted Embryonic Stem Cells Rescue Photoreceptor Cells From Degeneration in the RCS Rats

Ulrich Schraermeyer,1 Gabriele Thumann,1 Thomas Luther,1 Norbert Kociok,1 Stefan Arnhold,2 Klaus Kruttwig,2 Christian Andressen,2 Klaus Addicks,2 and Karl Ulrich Bartz-Schmidt1

1Department of Vitreoretinal Surgery and 2Department of Anatomy, University of Cologne, Joseph Stelzmann Str. 9, 50931 Cologne, Germany

The Royal College of Surgeons (RCS) rat is an animal model for retinal degeneration such as the age-related macular degeneration. The RCS rat undergoes a progressive retinal degeneration during the early postnatal period. A potential treatment to prevent this retinal degeneration is the transplantation into the subretinal space of cells that would replace functions of the degenerating retinal pigment epithelium (RPE) cells or may form neurotrophic factors. In this study we have investigated the potential of subretinally transplanted embryonic stem cells to prevent the genetically determined photoreceptor cell degeneration in the RCS rat. Embryonic stem cells from the inner cell mass of the mouse blastocyst were allowed to differentiate to neural precursor cells in vitro and were then transplanted into the subretinal space of 20-day-old RCS rats. Transplanted and sham-operated rats were sacrificed 2 months following cell transplantation. The eyes were enucleated and photoreceptor degeneration was quantified by analyzing and determining the thickness of the outer nuclear layer by light and electron microscopy. In the eyes transplanted with embryonic cells up to 8 rows of photoreceptor cell nuclei were observed, whereas in nontreated control eyes the outer nuclear layer had degenerated completely. Transplantation of embryonic stem cells appears to delay photoreceptor cell degeneration in RCS rats.

Key words: Electron microscopy; RCS rat; Stem cell; Transplantation

Address correspondence to Dr. Ulrich Schraermeyer, Department of Vitreoretinal Surgery, University of Cologne, Joseph Stelzmann Str. 9, 50931 Cologne, Germany. Tel: 49-221-4785730; Fax: 49-2131-745746; E-mail: U.Schraemeyer@uni-koeln.de

Cell Transplantation, Vol. 10, pp. 681-689
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Lipid-Mediated In Vivo Gene Transfer Replaces the Loss of Choline Acetyltransferase Activity After Unilateral Fimbria-Fornix Aspiration

Carla Weis, Walter A. Kaufmann, and Christian Humpel

Laboratory of Psychiatry, Clinic of Psychiatry, University Hospital Innsbruck, Austria

In Alzheimer's disease cholinergic neurons degenerate, resulting in loss of hippocampal acetylcholine. The fimbria-fornix aspiration is a well-known animal model mimicking hippocampal cholinergic deficiency. The aim of the present study was to use in vivo lipid-mediated gene transfer to introduce an expression vector coding for the acetylcholine synthesizing enzyme choline acetyltransferase into the hippocampus to replace the loss of enzyme activity after unilateral fimbria-fornix aspiration. Our data show that the lipid FuGene is useful to transfer DNA in vitro into 3T3 fibroblasts, C6 glioma cells, and primary astroglia and to express the respective enzyme. Lipid-mediated gene transfer in vivo resulted in a marked but transient expression of green fluorescent protein below the injection site peaking 5 days after the injection. Unilateral fimbria-fornix aspiration led to a marked reduction in the activity of choline acetyltransferase in the hippocampus, which was completely replaced 5 days after lipid-mediated gene transfer of the choline acetyltransferase vector. In conclusion, our data provide evidence that lipid-mediated gene transfer using FuGene is a useful tool to replace loss of choline acetyltranseferase activity in the hippocampus after fimbria-fornix aspiration; however, the lack of good gene transfer efficiency and the transient nature of expression limit its use for clinical applications.

Key words: Acetylcholine; Alzheimer's disease; Choline acetyltransferase; FuGene; Nonviral; Gene therapy

Address correspondence to Dr. Christian Humpel, Clinic of Psychiatry, Anichstr. 35, A-6020 Innsbruck, Austria. Tel: +43-512-504-3712; Fax: +43-512-504-3713; E-mail: christian.humpel@uibk.ac.at

Cell Transplantation, Vol. 10, pp. 689-696
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Automated Method for Isolation of Adrenal Medullary Chromaffin Cells From Neonatal Porcine Glands

Caterina Vizzardelli, Elizabeth D. Potter, Thierry Berney, Antonello Pileggi, Luca Inverardi, Camillo Ricordi, and Jacqueline Sagen

Diabetes Research Institute and The Miami Project to Cure Paralysis, University of Miami School of Medicine, Miami, FL, 33136

An automated method for the isolation of neonatal porcine adrenal chromaffin cells is described. Adrenal chromaffin cells are potentially useful for therapeutic transplantation, but current isolation methodology suffers from labor intensiveness and variability in yield and viability due to imprecision of manual techniques, enzyme purity, and gland age and species. The described approach utilizes an adaptation of an automated procedure previously described for isolation of pancreatic islets. Results from neonatal porcine adrenal glands revealed consistent cell yields with high (approximately 99%) viability. Catecholamine assays showed that the resultant cultures continue to produce and secrete norepinephrine and epinephrine. Immunocytochemical analysis indicated that the majority of cells in the preparation are chromaffin cells and adrenal cortical cells. The procedure meets the following requirements: 1) minimal traumatic action on the adrenal chromaffin cells, 2) continuous digestion in which the adrenal cells that are progressively liberated can be saved from further mechanical action, 3) minimal human intervention in the digestion process, and 4) high yield and viability of the isolated adrenal chromaffin cells.

Key words: Adrenal gland; Chromaffin cells; Catecholamines; Neural transplantation; Isolation; Automated methodology

Address correspondence to Jacqueline Sagen, Ph.D., The Miami Project to Cure Paralysis, University of Miami School of Medicine, Lois Pope LIFE Center, 1095 NW 14th Terrace (R-48), Miami, FL 33136. Tel: (305) 243-5618; Fax: (305) 243-6017; E-mail: jsagen@miami.edu

Cell Transplantation, Vol. 10, pp. 697-708
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Nonviral Transfection of Intact Pancreatic Islets

J. R. T. Lakey,1 A. T. L. Young,1 D. Pardue,2 S. Calvin,2 T. E. Albertson,2 L. Jacobson,2 and T. J. Cavanagh2

1Department of Surgery, Surgical-Medical Research Institute, University of Alberta, Edmonton, Canada T6G 2N8
2Roche Molecular Biochemicals, Indianapolis, IN

Ex vivo gene transfer offers a potential means to introduce genes into cells, which may play an important role in preventing graft rejection and inducing graft tolerance. This study examined the efficiency and toxicity of several lipid-based transfection reagents (LipofectAMINE, DOTAP, and DOSPER) in intact pancreatic islets. Isolated islets were transfected with a pCMV-b-galactosidase plasmid using several DNA/liposome ratios (1:12) of liposomes (3-72 ml) and DNA (3 and 6 mg). Transfection efficiency was quantified by microscopic evaluation of b-galactosidase gene expression in whole intact islets. Functionality of the transfected islets was measured by insulin response to glucose solutions. All transfection reagents evaluated in this study transfected cells within the islets. As expected, untransfected controls and transfected islets with DNA alone did not express b-gal. The highest transfection efficiency and functional viability were obtained following a 48-h incubation after exposure to the transfection mixtures as follows: 3 ml DNA and 18 ml DOTAP/ml (1:6 ratio), 6 mg DNA and 12 ml DOSPER/ml (1:2 ratio), or 6 mg DNA and 12 ml Lipofect-AMINE/ml (1:2 ratio). The highest rate of transfected cells per islet was obtained using DOTAP. In vitro functionality was not significantly different between DOTAP and nontreated controls. However, optimal transfection efficiency doses of LipofectAMINE and DOSPER significantly reduced the stimulated insulin response of the transfected islets (p < 0.05, ANOVA). The calculated stimulation index (SI) was 7.8 ± 0.6 (mean ± SEM) for DOTAP-transfected islets compared with 8.4 ± 0.5 for nontransfected control islets (p = ns). The SI of DOSPER- and LipofectAMINE-transfected islets was significantly lower (6.1 ± 0.5 and 3.4 ± 0.5, respectively, p < 0.05). Lipid-based transfection using DOTAP at a DNA/lipid ratio of 1:6 provides an effective means of ex vivo gene delivery without compromising in vitro functionality of the transfected islets.

Key words: Islets; Liposomes; Transfection; Diabetes; Transplantation

Address correspondence to Jonathan R. T. Lakey, Ph.D., Assistant Professor of Surgery, Surgical-Medical Research Institute, University of Alberta, 1074 Dentistry/Pharmacy Building, Edmonton, Alberta T6G 2N8 Canada. Tel: (780) 492-3077; Fax: (780) 492-1627; E-mail: jonathan.lakey@ualberta.ca

Cell Transplantation, Vol. 10, pp. 709-716
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Characterization of an Important Enzymatic Component in Collagenase That Is Essential for the Effective Digestion of the Human and Porcine Pancreas

Ruo L. Chen and Roger F. L. James

Department of Surgery, University of Leicester, RKB, Royal Infirmary, Leicester LE2 7LX, UK

Recent clinical results from Edmonton have demonstrated the feasibility of achieving normoglycemia in type I diabetic patients by islet transplantation. One of the key issues in obtaining this success was transplanting sufficient numbers of islets by sequential transplants. Although the development of semipurified endotoxin-free Clostridium histolyticum-derived collagenase (Liberase) has improved islet yields from the human pancreas, batch-to-batch variation and loss of activity with time still hampers progress in obtaining consistent islet preparations. In order to define key components of crude collagenase, a panel of monoclonal antibodies (McAbs) was raised against crude collagenase. Monoclonal antibodies were generated by fusions between splenocytes of BALB/c mice immunized with Boheringer P collagenase and the myeloma cell line NS-0. These monoclonal antibodies were used as probes to study molecular differences between effective and ineffective collagenase batches using Western blotting. Two monoclonal antibodies (LDS71 and LDS81) were raised and characterized as recognizing separate epitopes on a 125-kDa component. Western blotting indicated that the 125-kDa band was rapidly broken down by storage or by dialysis in the presence of dithiothreitol. However, this breakdown could be prevented by the addition of leupeptin (a protease inhibitor) to the dialysis buffer. On testing fractions at 5-min intervals from the "Ricordi" digestion circuit during porcine and human pancreas digestion, the 125-kDa component was rapidly broken down in relatively ineffective collagenase batches but in effective batches was present throughout the digestion process. The correlation between the presence of the 125-kDa band and effectiveness of pancreas digestion suggests that this may be a key component in the formulation of C. histolyticum collagenase.

Key words: Collagenase; Pancreas digestion; Monoclonal antibody; Western blotting

Address correspondence to Dr. R. F. L. James, Department of Surgery, Robert Kilpatrick Building, University of Leicester, Royal Infirmary, Leicester LE2 7LX, UK. Tel: 0044 116 252 3138; Fax: 0044 116 252 3179; E-mail: rj1@le.ac.uk

Cell Transplantation, Vol. 10, pp. 717-722
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Polyurethane Foam/Spheroid Culture System Using Human Hepatoblastoma Cell Line (Hep G2) as a Possible New Hybrid Artificial Liver

Yo-ichi Yamashita,1 Mitsuo Shimada,1 Eiji Tsujita,1 Shinji Tanaka,1 Hiroyuki Ijima,2 Kohji Nakazawa,2 Ryoichi Sakiyama,2 Junji Fukuda,2 Tadayoshi Ueda,3 Kazumori Funatsu,2 and Keizo Sugimachi1

1Department of Surgery and Science, Graduate School of Medical Sciences, and 2Department of Chemical Systems and Engineering, Graduate School of Engineering, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan
3Dainippon Pharmaceutical Co., Ltd., Enoki 33-94, Suita, Osaka 564-0053, Japan

The risk of xenozoonosis infections poses the greatest obstacle against the clinical application of hybrid artificial liver support system (HALSS). Primary human hepatocytes are an ideal source for HALSS, but the shortage of human livers available for hepatocyte isolation limits this modality. To resolve this issue, we used human hepatocytes with replication capacity (fetal hepatocytes, Hep G2, and Huh 7) in a polyurethane foam (PUF)/spheroid culture system in vitro, and analyzed liver functions such as ammonia removal and albumin synthesis capacity; results were compared to those of porcine hepatocytes. Human fetal hepatocytes, Hep G2, and Huh 7 formed spheroids spontaneously within 24 h in a PUF/spheroid culture system; ammonia removal activity (mmol/106 nuclei/h) was upregulated, as was albumin synthesis activity (mg/106 nuclei/day). In particular, Hep G2 spheroids demonstrated high ammonia removal and albumin synthesis activities: 85% of the ammonia removal activity and 171.7% of the albumin synthesis activity of porcine hepatocytes in the monolayer culture. These results indicate the possibility of the development of a multicapillary PUF (MC-PUF) packed-bed culture system of hepatocyte spheroids as a HALSS using Hep G2.

Key words: Hybrid artificial liver support system (HALSS); Human hepatocytes; Hep G2; Polyurethane foam (PUF)/spheroid culture system

Address correspondence to Yo-ichi Yamashita, M.D., Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Tel: 81-92-642-5469; Fax: 81-92-642-5482; E-mail: yamashi@surg2.med.kyushu-u.ac.jp

Cell Transplantation, Vol. 10, pp. 723-729
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

The Efficacy of Prevascularization by Basic FGF for Hepatocyte Transplantation Using Polymer Devices in Rats

Kohei Ogawa,1 Katsuhiro Asonuma,1 Yukihiro Inomata,1 Ildeok Kim,1 Yoshito Ikada,2 Yasuhiko Tabata,3 and Koichi Tanaka1

1Department of Transplantation and Immunology, Kyoto University, Kyoto, Japan
2Suzuka University of Medical Sciences, Mie, Japan
3Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan

This study used polymer devices implanted in rats to investigate the effect of prevascularization by basic fibroblast growth factor (bFGF) on hepatocyte transplantation (HTx). Lewis rats served as both donors and recipients. Polyvinyl alcohol (PVA) sponges with either hydrogel containing bFGF (bFGF group) or distilled water (control group) were implanted between the mesenteric leaves of recipient rats. Hepatotrophic stimulation was induced by a portacaval shunt and a 70% partial hepatectomy. After 1 week of prevascularization, hepatocytes harvested from the donor Lewis rats using a collagenase digestive method were injected into the sponges. Specimens were harvested at 2 weeks, 1 month, and 2 months after HTx. Histologic examination revealed that the control groups contained small numbers of hepatocytes restricted to the peripheral areas of the sponges. However, a large number of hepatocytes, including clusters, was found distributed uniformly in the bFGF group. In the bFGF group at 2 weeks, 1 month, and 2 months, the percentage of the sponge occupied by hepatocytes was 7.21 ± 2.64%, 6.98 ± 2.59%, and 5.58 ± 3.77%, respectively. The corresponding ratios for the control group were 0.40 ± 0.39%, 0.40 ± 0.40%, and 0.87 ± 1.51%. In addition, the mean number of new blood vessels in the bFGF group was significantly greater than that in the control group at 0 days, 2 weeks, and 1 month after HTx. These results suggest that bFGF strongly induced vascularization, which enabled a large number of hepatocytes to survive in the polymer devices.

Key words: Hepatocyte transplantation; Polymer devices; bFGF; Prevascularization

Address correspondence to Kohei Ogawa, M.D., Department of Transplantation and Immunology, Kyoto University, Shogoinkawahara-cho, Sakyo-ku, Kyoto, 606-8507, Japan. Tel: +81-75-751-4323; Fax: +81-75-751-4348; E-mail: kohei@kuhp.kyoto-u.ac.jp

Cell Transplantation, Vol. 10, pp. 731-738
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

The Effect of Coculture With Nonparenchymal Cells on Porcine Hepatocyte Function

Paul G. Gregory, Christopher K. Connolly, Bryan E. Gillis, and Susan J. Sullivan

Organogenesis Inc., Canton, MA 02021

Porcine hepatocytes are currently being investigated as a therapy for patients suffering from acute liver failure. Incorporating hepatocytes in an extracorporeal device that would stabilize a patient until transplantation or recovery could dramatically decrease the mortality rate associated with this disease. The ability to maximize hepatocyte function would contribute significantly to being able to provide the required cell mass in a device of reasonable size. Several approaches have been effective at increasing rat hepatocyte function in vitro, including coculture with nonparenchymal cells. In this study, we investigated the effect of the addition of 3T3 cells to porcine hepatocyte culture and found that while there was an increase in albumin secretion, there was little or no effect on urea synthesis or cytochrome P450 activity.

Key words: Porcine hepatocytes; Coculture; Differentiated function; Nonparenchymal cells

Address correspondence to S. J. Sullivan, Organogenesis, Inc., 150 Dan Road, Canton, MA 02021. Tel: (781) 401-1050; E-mail: ssullivan@organo.com

Cell Transplantation, Vol. 10, pp. 739-747
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Comparative Phenotype and Immunogenicity of Freshly Isolated and Immortalized Rat Hepatocytes

Vincent Kaulek,1,2 Philippe Saas,2 Eliane Alexandre,1 Helen Grant,3 Lysiane Richert,1,4 Daniel Jaeck,1 Pierre Tiberghien,2 Philippe Wolf,1 and Agnès Azimzadeh1

1Laboratoire de Chirurgie Expérimentale, Fondation Transplantation, 5 Avenue Molière, 67200 Strasbourg, France
2Laboratoire de Thérapeutique Immuno-moléculaire, EFS/EA 2284/Inserm E-0119, 1 Bd A Fleming, BP 1937, 25020 Besancon cedex, France
3University of Strathclyde, Bioengineering Unit, Wolfson Center, 106 Rottenrow, Glasgow 64 ONW, Scotland
4Faculté de Médecine et Pharmacie, Laboratoire de Biologie Cellulaire, Place Saint Jacques, 25030 Besancon, France

Immortalized hepatocytes are an attractive cell source for hepatocyte transplantation and gene transfer. We compared the phenotype and immunogenicity of freshly isolated (FIH) and immortalized (IMH) rat hepatocytes. Effect of culture and proinflammatory cytokines (TNF-\GK\a, IFN-g) was studied on phenotype. FIH were isolated by collagenase digestion. Two SV40 immortalized hepatocyte cell lines were tested (RH1 and P9). Immunophenotyping was performed by FACS analysis using anti-rat-specific antibodies. Immunogenicity was evaluated by a mixed lymphocyte hepatocyte reaction (MLHR). FIH suspension was an almost homogeneous parenchymal cell population with few (1-2%) CD8+ cells. FIH showed a positive staining for ICAM-1 (20-35%) and for Class I (RT1A, 30-60%) but no staining for Class II (RT1B). After 48 h of culture, the already ICAM-1-positive cells were more strongly stained and additionally 3.6% of the cells (possibly endothelial cells) were Class II positive. IMH showed a consistent expression of Class I (93-97%) and ICAM-1 (95-97%) but no expression of Class II. Culture of IMH for 48 h had no effect on Class II expression but increased ICAM-1 expression. Addition of TNF-a at 1000 UI/ml to cultures of FIH or IMH increased Class I and ICAM-1 expression whereas IFN-g (50 or 1000 UI/ml) had no evident effect. Hepatocyte immunogenicity, assessed in MLHR and appreciated by the stimulation index (SI) test/SI syngeneic control, was similar for IMH (RH1: 2.68 ± 0.89; P9: 2.37 ± 0.78) and FIH (2.52 ± 0.18). In conclusion, despite some quantitative immunophenotypic differences, FIH and IMH induced the same proliferation rate of allogeneic T lymphocytes. Thus, immortalized hepatocytes may constitute an appropriate cellular model to study the prevention of hepatocyte rejection by gene transfer.

Key words: Allotransplantation; Freshly isolated hepatocytes; Immortalized hepatocytes; MHC; Intercellular adhesion molecule-1; CD8

Address correspondence to Pr. Philippe Wolf, Hôpital de Hautepierre, Service de Chirurgie générale et de Transplantation multiorganes, Avenue Molière, 67098 Strasbourg Cedex, France. Tel: (33) 388 12 72 79; Fax: (33) 388 12 72 86; E-mail: Philippe.Wolf@chru-Strasbourg.fr

Cell Transplantation, Vol. 10, pp. 749-754
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Wound Dressings Alter the Colony-Forming Efficiency of Keratinocytes in Cultured Sheet Grafts

Lea Ann deGraffenried and R. Rivkah Isseroff

Department of Dermatology and Tissue Bioengineering Laboratory, Universityof California, Davis, Davis, CA 95616

Cultured keratinocyte grafts transplanted for skin wound repair are often affixed to a wound dressing to facilitate handling. In this study, the ability of five different types of wound dressings to support cell viability and maintain stem cell populations in the cultured grafts was determined. Postconfluent keratinocyte (NHK) sheets were attached to wound dressings for 24 h and then released by trypsinization. Cell viability was determined and NHKs were assessed for clonogenic capacity by colony-forming efficiency (CFE) assays. CFEs for NHKs exposed to a collagen-bonded, bilaminate membrane and a polyurethane film were significantly less than control. On the other hand, CFEs for NHKs exposed to a collagen/alginate dressing and to petrolatum-impregnated gauze were significantly greater than control. The choice of a wound dressing carrier has implications for maintaining long-term viability of the transplanted sheet of epithelium.

Key words: Keratinocyte transplantation; Colony-forming units assay; Stem cell; Biological dressings

Address correspondence to R. Rivkah Isseroff, M.D., Dermatology Research TB 192, University of California, Davis, One Shields Avenue, Davis, CA 95616. Tel: (530) 752-9767; Fax: (530) 752-9766; E-mail: rrisseroff@ucdavis.edu

Cell Transplantation, Vol. 10, pp. 755-763
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

In Vitro Expression of Cartilage-Specific Markers by Chondrocytes on a Biocompatible Hydrogel: Implications for Engineering Cartilage Tissue

Makarand Risbud,1,2 Jochen Ringe,1 Ramesh Bhonde,2 and Michael Sittinger1

1Tissue Engineering Laboratory, University Medical Centre, Charit&eacute;, Humboldt University of Berlin, Tucholskystrasse-2, 10117 Berlin, Germany
2Tissue Engineering and Banking Laboratory, National Centre for Cell Science, Ganeshkhind, Pune 411 007, India

Natural cartilage tissue has a limited self-regenerative capacity; thus, strategies to replenish the lost cartilage are desired in reconstructive and plastic surgery. Tissue-engineered cartilage using biodegradable polymeric scaffolds is one such approach gaining wide attention. We have earlier demonstrated the biocompatible nature and ability of chitosan-gelatin hydrogel to maintain differentiated populations of respiratory epithelial cells. The aim of the present study was to evaluate its suitability as a substratum for inducing chondrocyte growth and differentiation. Electron microscopic (SEM) analysis of freeze-dried hydrogels showed a highly porous morphology with interconnections as seen in cross section. Chondrocytes were observed to attach and exhibited a differentiated phenotype with proper cell-cell contact on three-dimensional freeze-dried hydrogels. When cultured on two-dimensional hydrogel films they showed higher growth rates (4-6%) compared with a polystyrene (TCPS) control until 6 days (p > 0.05), which slowed down after 10 days. Immunofluorescent microscopic studies revealed that chondrocytes on hydrogel films exhibited comparable expression of b1 integrin (CD29) to TCPS controls, indicating the ability of the hydrogel substrate to maintain normal expression of b1 integrin. RT-PCR analysis of chondrocytes grown on hydrogel films showed that chondrocytes express the mRNA for extracellular matrix proteins such as collagen type IIa1 (COL IIa1), COL III, COL IXa3. Expression of COL I was less prominent than COL II as indication of differentiation. Expression of COL X could not be detected, suggesting an absence of chondrocyte hypertrophy. Chondrocytes also showed weak mRNA expression of aggrecan, a cartilage-specific proteoglycan. All of these results point out the ability of the chitosan-gelatin hydrogel to induce the expression of mRNAs for cartilage-specific extracellular matrix proteins by nasal septal chondrocytes. This hydrogel needs to be further evaluated for its ability to support chondrocyte-specific marker expression to explore the possibility of forming a tissue resembling natural cartilage in vitro.

Key words: Chitosan; Gelatin; Hydrogel; Tissue engineering; Chondrocytes; Collagens

Address correspondence to PD Dr. Michael Sittinger, Tissue Engineering Laboratory, University Medical Centre, Charité, Humboldt University of Berlin, Tucholskystrasse-2, 10117 Berlin, Germany. Tel: 049 30 450 513 198; Fax: 049 30 450 513 943; E-mail: michael.sittinger@charite.de

Cell Transplantation, Vol. 10, pp. 765-771
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Locomotion of Human Skin Keratinocytes on Polystyrene, Fibrin, and Collagen Substrata and its Modification by Cell-to-Cell Contacts

Justyna Drukala,1 Laura Bandura,1 Kazimierz Cieslik,2 and Wlodzimierz Korohoda1

1Department of Cell Biology, The Jan Zurzycki Institute of Molecular Biology, Jagiellonian University, Kraków, Poland
2Ludwik Rydygier Speciality Hospital in Kraków, Department of Burns and Plastic Surgery, Poland

Epithelial wound repair assures the recovery of the epithelial barrier after wounding. During wound healing epithelial cells migrate to cover the wound surface. The presented experiments were carried out to compare the migration of human keratinocytes from primary and secondary culture on polystyrene, collagen, and fibrin glue used in clinical techniques. The images of migrating keratinocytes were recorded and analyzed using computer-aided methods. The results show that the character of the substrate strongly affects the speed and turning behavior of keratinocytes locomoting over it. The highest motile activity of human skin keratinocytes was found on fibrin glue substratum. It was found that locomotion of freely moving isolated cells was much faster than that of cell sheets. The autologous keratinocytes cultured in vitro were applied with fibrin glue to cover trophic wounds. The transplantation of human autologous keratinocyte suspension in fibrin glue upon long-lasting trophic wounds appeared to induce rapid and permanent wound healing.

Key words: Keratinocytes; Wound healing; Fibrin glue; Locomotion

Address correspondence to Justyna Drukala, Ph.D., Department of Cell Biology, The Jan Zurzycki Institute of Molecular Biology, Jagiellonian University, ul. Gronostajowa 7, 30-387 Kraków, Poland. Tel: +48-12-252-61-45; Fax: +48-12-252-69-02; E-mail: justyna@mol.uj.edu.pl