Regulation of cutaneous pigmentation by titration of human melanocytes in cultured skin substitutes grafted to athymic mice

Pigmentation of healed cultured skin substitutes in burn patients is frequently irregular and unpredictable which compromises solar protection and the patient's self-image. To address these morbidities, human fibroblasts were inoculated on a collagen-glycosaminoglycan substrate followed 1 day later by the addition of keratinocytes at 1.1 x 10(6)/cm2 combined with either 0, 1.1 x 10(2), 1.1 x 10(3), or 1.1 x 10(4) melanocytes/cm2. The skin substitutes were incubated in vitro for 3 weeks and grafted to athymic mice. In vitro, the number of L-Dopa-positive melanocytes in the skin substitutes increased proportionately to the number of melanocytes inoculated. The melanocytes localized to the basal epidermis when labeled for MEL-5. The skin substitutes with 1.1 x 10(4) melanocytes/cm2 were significantly darker than other groups in vitro by chromameter evaluation. By 12 weeks after grafting, the cultured skin ranged from no pigment in the control group, to 75% pigmented area in the 1.1 x 10(3) melanocytes/cm2 group, to complete pigmentation in the 1.1 x 10(4) melanocytes/cm2 group. In vivo, the mean chromameter values were significantly darker for the grafts with 1.1 x 10(3) and 1.1 x 10(4) melanocytes/cm2. These results suggest that complete restoration of cutaneous pigmentation can be accomplished by addition of between 0.1 and 1.0 x 10(4) melanocytes/cm2 to skin substitutes.     

Wound healing on athymic mice with engineered skin substitutes fabricated with keratinocytes harvested from an automated bioreactor

The Kerator is a computer controlled bioreactor for the automated culture and harvest of keratinocytes that can reduce labor and materials involved in the fabrication of engineered skin substitutes (ESS). Previous studies have shown that the Kerator is comparable to tissue culture flasks by keratinocyte confluence during culture, clonogenic potential of harvested keratinocytes and microanatomy, cell viability, and surface hydration of ESS fabricated with the harvested keratinocytes. In this study, the Kerator and tissue culture flasks were further compared by keratinocyte proliferation in vitro and wound healing after transplantation of ESS to athymic mice. The number of bromodeoxyuridine-positive keratinocytes in ESS fabricated with keratinocytes harvested from Kerator after 2 wk of in vitro maturation was 34 +/- 3 per high power field (hpf) (mean +/- SEM), which was not significantly different from that fabricated with keratinocytes harvested from flasks (34 +/- 1.5 per hpf). Percentage original wound area 6 wk after surgery of ESS fabricated with keratinocytes from the Kerator was 36% +/- 3.3%, which was not significantly different from that of ESS fabricated with keratinocytes from flasks (30% +/- 4.3%). In both cases, 78% (7 of 9) mice transplanted were positive for engraftment of human keratinocytes by direct immunofluorescence for HLA-ABC antigens. These results further confirm that the ESS fabricated with keratinocytes harvested from Kerator and flasks are equivalent in vitro and in vivo. Therefore, use of Kerator for large scale production of ESS can lead to increased availability at reduced cost while maintaining ESS quality for grafting.     

Reconstructed human skin produced in vitro and grafted on athymic mice

BACKGROUND: The best alternative to a split-thickness graft for the wound coverage of patients with extensive burns should be in vitro reconstructed autologous skin made of both dermis and epidermis and devoid of exogenous extracellular matrix proteins and synthetic material. We have designed such a reconstructed human skin (rHS) and present here its first in vivo grafting on athymic mice. METHODS: The rHS was made by culturing newborn or adult keratinocytes on superimposed fibrous sheets obtained after culturing human fibroblasts with ascorbic acid. Ten days after keratinocyte seeding, reconstructed skins were either cultured at the air-liquid interface or grafted on athymic mice. We present the macroscopic, histologic, and phenotypic properties of such tissues in vitro and in vivo after grafting on nude mice. RESULTS: After maturation in vitro, the reconstructed skin exhibited a well-developed human epidermis that expressed differentiated markers and basement membrane proteins. Four days after grafting, a complete take of all grafts was obtained. Histological analysis revealed that the newly generated epidermis of newborn rHS was thicker than that of adult rHS after 4 days but similar 21 days after grafting. The basement membrane components (bullous pemphigoid antigens, laminin, and type IV and VII collagens) were detected at the dermo-epidermal junction, showing a continuous line 4 days after grafting. Ultrastructural studies revealed that the basement membrane was continuous and well organized 21 days after transplantation. The macroscopic aspect of the reconstructed skin revealed a resistant, supple, and elastic tissue. Elastin staining and elastic fibers were detected as a complex network in the rHS that contributes to the good elasticity of this new reconstructed tissue. CONCLUSIONS: This new rHS model gives supple and easy to handle skins while demonstrating an adequate wound healing on mice. These results are promising for the development of this skin substitute for permanent coverage of burn wounds.     

Fibroblasts improve performance of cultured composite skin substitutes on athymic mice

BACKGROUND: This study investigated the impact of adding human fibroblasts to a cultured composite skin substitute model of cultured human keratinocytes and acellular human dermis. METHODS: Skin substitutes were prepared by seeding human keratinocytes on the papillary side of acellular dermis with or without seeding fibroblasts on the reticular side. Performance of the grafts was compared both in vitro by histology and in vivo on surgically created full-thickness wounds on athymic mice. Graft size and contraction were measured and immunohistochemical stains were done to reveal vascularization. RESULTS: Skin substitutes with fibroblasts formed thicker epidermis than skin substitutes without fibroblasts. When transplanted onto athymic mice, skin substitutes with fibroblasts maintained their original size with only 2% contraction. In contrast, skin substitutes without fibroblasts showed 29% contraction. Vascular basement membrane specific mouse CD31staining and endothelial cell specific mouse collagen type IV staining revealed vascularization as early as 1 week posttransplant in grafts with fibroblasts, and was significantly higher than grafts without fibroblasts at 2 weeks. CONCLUSIONS: Addition of fibroblasts to keratinocyte based composite skin substitutes improves epidermis formation, enhances vascularization and reduces contraction.     

Effective management of microbial contamination in cultured skin substitutes after grafting to athymic mice.

Cultured skin substitutes have become therapeutic alternatives for treatment of acute and chronic skin wounds, but all models of these substitutes are avascular and susceptible to microbial destruction during vascularization. To develop a practical management protocol for increased survival of skin substitutes, experimental wounds were contaminated with Pseudomonas aeruginosa and treated with a formulation of noncytotoxic antimicrobial agents (polymyxin B, neomycin, ciprofloxacin, mupirocin, amphotericin B) in a nutrient medium (vehicle). Cultured skin substitutes consisting of human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan sponges were grafted to 2 x 2 cm full-thickness wounds on athymic mice that were contaminated with the strain SBI-N of P. aeruginosa at 1 x 10(4), 1 x 10(5), and 1 x 10(6) organisms/wound. Experimental wounds were irrigated with 1 ml/day topical antimicrobial solution for 10 days, and controls received vehicle only. Two, three, and four weeks after grafting, wounds were traced and swabbed for microbial culture and areas were measured with planimetry. At 4 weeks, biopsy samples were scored histochemically for immunoreactivity to HLA antigens. Data analysis by chi-square, analysis of variance, and Tukey's test shows that treatment of contaminated wounds with noncytotoxic topical antimicrobials is associated with an increased area of healed wounds, positive detection of HLA antigens, and negative cultures for P. aeruginosa. These results show that microbial contamination of cultured skin substitutes on full-thickness wounds may be managed effectively during graft vascularization. However, this formulation of antimicrobial agents is not currently approved for human use and is investigational only. Effective management of microbial contamination suggests that clinical efficacy of avascular tissue analogs may be increased by local application of noncytotoxic antimicrobial agents.     

Epidermal lipid metabolism of cultured skin substitutes during healing of full-thickness wounds in athymic mice

Cultured epidermal keratinocytes provide an abundant supply of biologic material for wound treatment. Because restoration of barrier function is a definitive criterion for efficacy of wound closure and depends on the lipids present in the epidermis, we analyzed lipid composition of the epidermis in cultured skin substitutes in vitro and after grafting to athymic mice. The cultured skin substitutes were prepared from human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan substrates. After 14 days of incubation, cultured skin substitutes were grafted orthotopically onto full-thickness wounds in athymic mice. Samples for lipid analysis were collected after 14 and 34 days of in vitro incubation, and 3 weeks and 4 months after grafting. Both in vitro samples show disproportions in epidermal lipid profile as compared with the native human epidermis, i.e., a low amount of phospholipids (indicating imbalance in proliferation and differentiation); a large excess of triglycerides (storage lipids); and low levels of free fatty acids, gluco-sphingolipids, cholesterol sulfate, and ceramides-suggesting abnormal composition of stratum corneum barrier lipids. Fatty acid analysis of cultured skin substitutes in vitro revealed insufficient uptake of linoleic acid, which resulted in increased synthesis of and substitution with monounsaturated fatty acids, mainly oleic acid. These abnormalities were partially corrected by 3 weeks after grafting; and 4 months after grafting, all epidermal lipids, with some minor exceptions, were synthesized in proportions very similar to human epidermis. Results of this study show that grafting of cultured skin substitutes to a physiologic host permits the recovery of lipid in proportion to that required for barrier formation in normal human epidermis.     

人胚胎成纤维细胞作为组织工程皮肤种子细胞的可行性研究

目的：评估人胚胎成纤维细胞（human embryonic fibroblasts,HEF）作为组织工程皮肤种子细胞的可行性和优越性。方法：取人流产胚胎皮肤、正常儿童皮肤,相同条件下分离培养成纤维细胞,比较两组细胞镜下、超微结构以及增殖特性,异体淋巴细胞混合实验对比其抗原性。以第三代细胞复合鼠尾胶原构建三维培养,ELASA法分别测定两组三维构建培养液中IL-6,TGF-β1含量。结果：与普通成纤维细胞相比,胚胎成纤维细胞扩增后具有更好的细胞形态和功能,生长速度快,分裂指数高,几乎不刺激异体淋巴细胞增殖。在鼠尾胶原支架中成纤维细胞生长状态良好,并且具有一定的组织强度。胎儿成纤维细胞组培养液中的TGF-β1和IL-6在各个时相上分别显著低于和高于普通成纤维细胞组。结论：胚胎成纤维细胞是组织工程皮肤较理想的种子细胞。     

Incubation of cultured skin substitutes in reduced humidity promotes cornification in vitro and stable engraftment in athymic mice

Cultured skin substitutes have been used successfully for adjunctive treatment of excised burns and chronic skin wounds. However, limitations inherent to all models of cultured skin include deficient barrier function in vitro, and delayed keratinization after grafting in comparison to native skin autografts. Experimental conditions for incubation of skin substitutes were tested to stimulate barrier development before grafting, and measure responses in function and stability after grafting. Cultured skin substitutes consisted of human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan biopolymer substrates. Parallel cultured skin substitutes were incubated at the air-liquid interface in ambient (48-61%) or saturated (79-91%) relative humidity, and grafted to athymic mice on culture day 14. Additional cultured skin substitutes were incubated in the experimental conditions for a total of 28 days. Cadaveric human skin and acellular biopolymer substrates served as controls. Epidermal barrier was evaluated as the change in surface hydration by surface electrical capacitance with the NOVA Dermal Phase Meter. Cultured skin substitutes and cadaveric skin incubated in ambient humidity had lower baseline surface electrical capacitance and less change in surface electrical capacitance than parallel samples incubated in saturated humidity at all time points in vitro. Data from healing cultured skin substitutes at 2, 4, 8 and 12 weeks after grafting showed an earlier return to hydration levels comparable to native human skin, and more stable engraftment for skin substitutes from ambient humidity. The data indicate that cultured skin substitutes in ambient humidity have lower surface electrical capacitance and greater stability in vitro, and that they reform epidermal barrier more rapidly after grafting than cultured skin substitutes in saturated humidity. These results suggest that restoration of functional epidermis by cultured skin substitutes is stimulated by incubation in reduced humidity in vitro.     

Use of a composite skin graft composed of cultured human keratinocytes and fibroblasts and a collagen-GAG matrix to cover full-thickness wounds on athymic mice

In patients with extensive full-thickness burns, wound coverage may be accelerated if skin can be expanded to produce a skin replacement that reproducibly supplies blood to the wound and has good structural qualities. In addition, development of skin replacements may benefit patients who require reconstruction or replacement of large areas of abnormal skin. We have developed a composite skin replacement composed of cultured human keratinocytes (HK) and fibroblasts. Cultured human fibroblasts are seeded into the interstices, and cultured HKs are applied to the surface of a matrix composed of type I collagen crosslinked with a glycosaminoglycan, which has a defined physical structure. After HKs reach confluence on the matrix surface, the composite grafts are placed on full-thickness wounds on the dorsum of athymic mice. Graft acceptance, confirmed by positive staining with antibodies specific for human HLA-ABC antigens on HKs, is approximately 90%. A defined skin structure is present histologically by day 10 after grafting, with a differentiated epithelium and a subepidermal layer densely populated by fibroblasts and capillaries without evidence of inflammation. Fluorescent light microscopy to identify laminin and type IV collagen and electron microscopy confirm the presence of basement membrane components by 10 days after grafting. Attachment of the graft to the wound is similar with and without the addition of human basic fibroblast growth factor, a potent angiogenic agent, to the skin replacement before graft placement on wounds.     

Cultured human keratinocytes as a single cell suspension in fibrin glue combined with preserved dermal grafts enhance skin reconstitution in athymic mice full-thickness wounds

Cultured human keratinocytes as a single cell suspension in fibrin glue combined with preserved dermal grafts enhance skin reconstitution in athymic mice full-thickness wounds. The technique of transplanting cultured human keratinocytes suspended as single cells in a fibrin-glue matrix (KFGS) has been recently developed to overcome common disadvantages of standard cultured epidermal sheet grafts. The combination of this method with glycerolized (nonvital) xenograft overlays in standardized nude mice full-thickness wounds, as compared to KFGS alone or controls with no grafts, showed enhancement of epithelial regeneration in terms of epithelial thickness and diminished wound contraction during the 6-week follow-up. Total scar thickness was increased after the combined KFGS/xenograft technique. The time taken to complete wound reepithelialization was similar in the two groups. Reconstitution of the dermo-epidermal junction zone as shown by electron microscopy and immunohistochemistry was enhanced by the KFGS+xenograft technique, showing structures resembling rete ridges 6 weeks postoperatively. The combined KFGS/xenograft technique is able to transfer proliferative single keratinocytes. The method simplifies the application when compared to conventional epithelial sheet grafting and reduces wound contraction when compared to pure keratinocyte grafting. Received: 15 October 1998 / / Accepted: 10 March 1999     

Wound-healing factors secreted by epidermal keratinocytes and dermal fibroblasts in skin substitutes

Full-skin substitutes, epidermal substitutes, and dermal substitutes are currently being used to heal deep burns and chronic ulcers. In this study, we investigated which wound-healing mediators are released from these constructs and whether keratinocyte-fibroblast interactions are involved. Autologous skin substitutes were constructed from human keratinocytes, fibroblasts, and acellular donor dermis. Full-thickness skin was used to represent an autograft. Secretion of wound-healing mediators was investigated by means of protein array, enzyme-linked immunosorbent assay, neutralizing antibodies, and conditioned culture supernatants. Full-skin substitutes and autografts produce high amounts of inflammatory/angiogenic mediators (IL-6, CCL2, CXCL1, CXCL8, and sST2). Epidermal and dermal substitutes produced less of these proteins. Epidermal-derived proinflammatory cytokines interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) were found to mediate synergistically the secretion of these wound-healing mediators (with the exception of sST2) from fibroblasts in dermal substitutes. The secretion of proinflammatory cytokines (IL-1alpha, TNF-alpha), chemokine/mitogen (CCL5) and angiogenic factor (vascular endothelial growth factor) by epidermal substitutes and tissue remodeling factors (tissue inhibitor of metalloproteinase-2, hepatocyte growth factor) by dermal substitutes was not influenced by keratinocyte-fibroblast interactions. The full-skin substitute has a greater potential to stimulate wound healing than epidermal or dermal substitutes. Both epidermal-derived IL-1alpha and TNF-alpha are required to trigger the release of dermal-derived inflammatory/angiogenic mediators from skin substitutes.     

Modeling the effects of treating diabetic wounds with engineered skin substitutes

In this paper, a novel mathematical model of wound healing in both normal and diabetic cases is presented, focusing upon the effects of adding two currently available commercial engineered skin substitute therapies to the wound (Apligraf) and Dermagraft). Our work extends a previously developed model, which considers inflammatory and repair macrophage dynamics in normal and diabetic wound healing. Here, we extend the model to include equations for platelet-derived growth factor concentration, fibroblast density, collagen density, and hyaluronan concentration. This enables us to examine the variation of these components in both normal and diabetic wound healing cases, and to model the treatment protocols of these therapies. Within the context of our model, we find that the key component to successful healing in diabetic wounds is hyaluronan and that the therapies work by increasing the amount of hyaluronan available in the wound environment. The time-to-healing results correlate with those observed in clinical trials and the model goes some way to establishing an understanding of why diabetic wounds do not heal, and how these treatments affect the diabetic wound environment to promote wound closure.     

Genetic modification of cultured skin substitutes by transduction of human keratinocytes and fibroblasts with platelet-derived growth factor-A

Gene therapy promises the potential for improved treatment of cutaneous wounds. This study evaluated whether genetically modified cultured skin substitutes can act as vehicles for gene therapy in an athymic mouse model of wound healing. Human keratinocytes and fibroblasts were genetically engineered by retroviral transduction to overexpress human platelet-derived growth factor-A chain. Three types of skin substitutes were prepared from collagen-glycosaminoglycan substrates populated with fibroblasts and keratinocytes: HF-/HK-, containing both unmodified fibroblasts and keratinocytes; HF-/HK+, containing unmodified fibroblasts and modified keratinocytes; and HF+/HK-, containing modified fibroblasts and unmodified keratinocytes. Skin substitutes were cultured for two weeks before grafting to full-thickness wounds on athymic mice. The modified skin substitutes secreted significantly elevated levels of platelet-derived growth factor throughout the culture period. Expression of retroviral platelet-derived growth factor-A mRNA was maintained after grafting to mice, and was detected in all HF-/HK+ grafts and one HF+/HK- graft at two weeks after surgery. Although no differences were seen between control and modified grafts, the results suggest that genetically modified cultured skin substitutes can be a feasible mechanism for cutaneous gene therapy. The cultured skin model used for these studies has advantages over other skin analogs containing only epidermal cells; because it contains both fibroblasts and keratinocytes, it therefore offers greater opportunities for genetic modification and potential modulation of wound healing.     

Expression of human beta-defensins HBD-1, HBD-2, and HBD-3 in cultured keratinocytes and skin substitutes

Defensins are effector molecules of the innate host defense system with antimicrobial activity against a variety of pathogens, including microorganisms commonly found in burn units. beta-Defensins are variably expressed in the epithelia of skin and other organs. Cultured skin substitutes (CSS) grafted to burn wounds lack a vascular plexus and are therefore more susceptible to microbial contamination than split thickness skin autograft. To investigate whether beta-defensins can contribute to host defense in CSS, we examined expression of human beta-defensins HBD-1, HBD-2, and HBD-3 in cultured keratinocytes and CSS from uninjured donors and burn patients. HBD-1 was expressed in all keratinocyte strains analyzed. HBD-2 expression in keratinocyte monolayers was highly variable but did not correlate with burn injury. HBD-3 was expressed at variable levels in all but one keratinocyte strain. CSS were prepared from two donors that lacked expression of HBD-2 in keratinocyte monolayers. All three genes were readily detected in CSS from both donors, suggesting up-regulation of HBD-2 and HBD-3. In sections of CSS, HBD-1, HBD-2, and HBD-3 proteins were localized to distinct epidermal regions. We conclude that beta-defensins can potentially contribute to innate immunity in CSS, but their levels may be too low to prevent contamination after grafting.     

毛乳头细胞促进组织工程皮肤血管化的实验研究

目的探讨毛乳头细胞对表皮干细胞与同种异体脱细胞真皮基质构建的组织工程皮肤血管化的影响。方法取门诊包皮环切术患者皮肤,用于表皮细胞培养;取孕19、20周引产胎儿头部皮肤,用于毛乳头细胞培养;患者及家属均知情同意。以中性蛋白酶联合胰蛋白酶消化、分离表皮细胞,IV型胶原黏附法分选表皮于细胞;以Ⅰ型胶原酶消化法分离毛乳头,常规成纤维细胞培养液培养。以同种异体脱细胞真皮基质为支架,在真皮基质的真皮乳头侧种植毛乳头细胞,基底膜侧种植表皮干细胞构建实验组组织工程皮肤替代物;对照组组织工程皮肤替代物不种植毛乳头细胞。取6～8周龄BALB/C-nu裸鼠60只,随机分成实验组和对照组（n=30）;实验动物背部制造1 cm×1 cm大小全层皮肤缺损模型,将两组组织工程皮肤替代物移植修复创面,2周后观察移植皮肤成活率。术后2、4周,取移植皮肤替代物标本,行HE及免疫组织化学染色,观察移植皮肤替代物CD31表达水平,并计算两组标本血管密度。结果Ⅳ型胶原分选后表皮细胞同时表达角蛋白19、β₁整合素,提示为表皮干细胞。由毛乳头培养出的细胞表达α平滑肌肌动蛋白,提示为毛乳头细胞。动物移植术后2周,实验组移植皮肤成活率为93.3%（28/30）,对照组为80.0%（24/30）,比较差异有统计学意义（χ²=2.31,P=-0.00）。HE染色观察示实验组表皮层达12层,表皮细胞体积较大;对照组表皮层达4～6层,表皮细胞体积较小。实验组术后2、4周,血管密度分别为（38.56±2.49）个/mm2和（49.12±2.39）个/mm²,对照组分别为（25.16±3.73）个/mm²和（36.26±3.24）个/mm²,两组比较差异均有统计学意义（P<0.01）。结论毛乳头细胞可促进移植皮肤替代物血管形成,利于表皮层重建,提高组织工程皮肤移植成活率。     

含内皮祖细胞的组织工程皮肤体外构建及裸鼠移植研究

目的构建含内皮祖细胞（EPC）和成纤维细胞的组织工程复合皮，初步研究EPC在组织工程皮肤移植中促血管发生的作用。方法采用免疫磁珠法从人脐血中分离培养CD133＋血管内皮祖细胞，以聚羟基乙酸（PGA）为真皮基质，接种EPC和成纤维细胞，其上覆盖表皮细胞膜片，构建组织工程复合皮，覆盖裸鼠背部全层皮肤缺损创面。结果EPC和成纤维细胞能均匀贴附于PGA支架纤维材料上，并逐渐伸展为梭形或多极状。裸鼠移植实验表职，实验组创面愈合早于对照组，可见EPC参与大量新生小血管形成，真皮支架5周完全降解。结论EPC参与血管重建，具有促进血管新生，加速缺血组织血管化的作用。应用PGA＋纤维蛋白凝胶，制备含EPC和成纤维细胞的活性真皮替代物，具有良好的生物相容性、降解特性。     

Heterotransplantation of human transitional cell carcinoma in athymic mice.

Human bladder tumors were obtained and transplanted into nude mice or other control animals. Tumor measurements, growth rate and selected histological studies were completed. No correlation between the growth of the tumor in the nude mouse and the clinical course of histologic tumor appearance in the host was detected. Metastases were not found. This feature has been noted generally to be uncommon in the nude mice model with some other human tumors. Despite careful technique tumor growth was achieved in only 40% of the appropriate experiments. The factors responsible for this variability and different growth rate in the nude mouse require further assessment before the results of other experimental treatments can be evaluated.     

Heterotransplantation of human craniopharyngiomas in athymic "nude" mice.

Surgical biopsies from 2 human craniopharyngiomas were transplanted subcutaneously into 6 athymic "nude" mice. Morphologically characteristic craniopharyngiomas grew in 5 of these animals. In 4 animals growth was sufficient to allow transplantation into a second generation of animals. In all, 11 craniopharyngiomas were present at autopsy in the 14 animals into which the tumors had been transplanted. The tumors that grew in the animals had the same adamantinomatous architecture, epithelial nests, and keratinized nodules that were present in the original surgical sample and that are characteristic of human craniopharyngiomas. It may be possible to study growth characteristics and therapeutic sensitivities of human craniopharygniomas growing in "nude" mice.     

Growth and hormonal content of human fetal pancreas passaged in athymic mice

While the usefulness of the human fetal pancreas transplanted into diabetic humans has yet to be realized, its transplantation into nude mice has revealed some of its potential. In this animal the organ grows in size, during which time differentiation of its endocrine component and maturation of the insulinogenic response to glucose occurs. The results reported in this article expand these results by providing data on the weight and both the insulin and glucagon content of these passaged organs. Human fetal pancreata of gestational age 14-19 wk were implanted subcutaneously (s.c.) in nude mice and maintained in vivo for 5-54 wk (absolute age 19-68 wk). The implants were then removed and their insulin and glucagon content determined. Both the weight of these implants and their insulin content were positively correlated with the absolute age of the tissue (weight: r = 0.61, P less than 0.001; insulin content: r = 0.62, P = 0.0003). The glucagon content bore no relationship to the age. The maximum level of insulin extracted from 30 passaged human fetal pancreata was 0.8 U, a level far below the daily insulin requirements in adult humans. It is suggested that an explanation for this is the site of transplantation used. To compare the s.c. site with the renal subcapsular space, the explants of four fetal pancreata were evenly divided between these two sites. After 11-13 wk, the implants were removed. Those beneath the renal capsule were larger and contained a greater amount of insulin and glucagon than those transplanted s.c.(ABSTRACT TRUNCATED AT 250 WORDS)