Development of a stable chemically defined surface for the culture of human keratinocytes under serum-free conditions for clinical use

Within the field of tissue engineering there is a need to develop new approaches to achieve effective wound closure in patients with extensive skin loss or chronic ulcers. This article exploits the well-known interdependency of epithelial keratinocytes and stromal fibroblasts in conjunction with plasma surface technology. The aim was to produce a chemically defined surface, which with the aid of a feeder layer of lethally irradiated dermal fibroblasts would improve the attachment and proliferation of the keratinocyte cell from which subconfluent cells can be transferred to wound bed models. Plasma copolymers of acrylic acid/octa-1,7-diene have been prepared and characterized by X-ray photoelectron spectroscopy. The fibroblasts and keratinocytes were cultured on plasma polymer-coated 24-well plates. Cell attachment and proliferation were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-eluted stain assay (MTT-ESTA) and DNA assay. Attachment and proliferation of both cell types on plasma polymer surfaces were compared with tissue culture plastic and collagen I, plus a negative control of a pure hydrocarbon layer. A pure acrylic acid surface, fabricated at a power of 10 W and containing 9.2% carboxylate groups, was found to promote both fibroblast and keratinocyte attachment and proliferation and permit the serum-free coculture of keratinocytes and irradiated fibroblasts.     

Fibroblast sheets enable epithelialization of sounds that do not support keratinocyte migration

Keratinocyte migration over the wound bed is the single most important parameter for wound epithelialization. Therefore, improvement of the wound bed matrix holds considerable promise for the shortening of hospitalization time in patients with ulcers, burns, and chronic wounds. We investigated wound epithelialization in athymic mice in the presence or absence of a sheet of cultured human fibroblasts. The physiology of keratinocyte growth on fibroblast sheets was investigated in tissue culture using histology, immunofluorescence, and electron microscopy. Keratinocytes from human skin explants were unable to attach or migrate on full-thickness dorsal wounds of athymic mice. Placement of a fibroblast-seeded polyglactin mesh on the wounds resulted in dramatically increased keratinocyte outgrowth. Similarly, human keratinocytes showed good outgrowth on fibroblast sheets at the air/liquid interface in tissue culture. Outgrowth was correlated inversely with fibroblast viability, indicating that the observed effect was due to the complex extracellular matrix secreted by the fibroblasts and matrix-bound growth factors rather than ongoing growth factor release. Collagen IV, a promoter of keratinocyte migration, was found to be abundant in the fibroblast-derived matrix. This study demonstrates that wounds which are unable to support keratinocyte migration can undergo epithelialization if a conducive substrate, supplying appropriate extracellular matrix and/or matrix-bound growth factors, is applied.     

Cultured human keratinocytes on type I collagen membranes to reconstitute the epidermis

The development of new techniques and modifications to overcome some of the disadvantages in cultured keratinocyte grafting has been motivated by several well-known drawbacks in the use of cultured epithelial autografts such as long culture periods, lack of adherence, difficulty in handling, lack of dermal substrates, and high costs. Two recent insights have influenced further research. On the one hand, it has been shown that the use of undifferentiated proliferative cells in fibrin glue suspensions is effective in epithelial reconstitution. On the other hand, the enzymatic release of cells from the culture surfaces is a critical step leading to at least temporary destruction of anchoring structures of the cultured cells. In this study, we tried to combine these two aspects in an attempt to modify common modalities of keratinocyte transplantation. To avoid dispase dissolving of the cultured cells, keratinocytes were seeded onto bovine collagen type I membranes without feeder layers and under serum-free culture conditions. Subconfluent monolayers of cultured human keratinocytes were transplanted as an upside-down graft on collagen membranes (keratinocyte collagen membrane grafts [KCMG], n = 12) after 3 days of culture or as membrane grafts alone (n = 12) onto standard nude mice full-thickness wounds. Fully differentiated epidermis was found at 21 days after grafting KCMG with persistence of human keratinocytes. This study demonstrates that upside-down grafts of undifferentiated monolayers of keratinocytes on non-cross-linked bovine type I collagen membranes do lead to an early reconstitution of multilayered squamous epithelium with enhanced wound healing compared to the control group. The upside down KCMG grafting technique is able to transfer actively proliferative keratinocytes and simplifies the application compared to conventional epithelial sheet grafting.     

The effect of chitin and chitosan on the proliferation of human skin fibroblasts and keratinocytes in vitro

The effects of chitin [(1 --> 4)-2-acetamido-2-deoxy-beta-D-glucan] and its partially deacetylated derivatives, chitosans, on the proliferation of human dermal fibroblasts and keratinocytes were examined in vitro. Chitosans with relatively high degrees of deacetylation strongly stimulated fibroblast proliferation while samples with lower levels of deacetylation showed less activity. Fraction, CL313A, a shorter chain length, 89% deacetylated chitosan chloride was further evaluated using cultures of fibroblasts derived from a range of human donors. Some fibroblast cultures produced a positive mitogenic response to CL313A treatment with proliferation rates being increased by approximately 50% over the control level at an initial concentration of 50 microg/ml, whilst others showed no stimulation of proliferation or even a slight inhibition (< 10%). The stimulatory effect on fibroblast proliferation required the presence of serum in the culture medium suggesting that the chitosan may be interacting with growth factors present in the serum and potentiating their effect. In contrast to the stimulatory effects on fibroblasts, fraction CL313A inhibited human keratinocyte mitogenesis with up to 40% inhibition of proliferation being observed at 50 microg/ml. In general highly deacetylated chitosans were more active than those with a lower degree of deacetylation. These data demonstrate that highly deacetylated chitosans can modulate human skin cell mitogenesis in vitro. Analysis of their effects on cells in culture may be useful as a screen for their potential activity in vivo as wound healing agents, although in the case of fibroblasts it is important to select appropriate strains of cells for use in the screen.     

Generation of human epidermal constructs on a collagen layer alone

Because engineered tissues are designed for clinical applications in humans, a major problem is the contamination of cocultures and tissues by allogenic molecules used to grow stem cells in vitro. The protocols that are commonly applied to generate epidermal equivalents in vitro require the use of irradiated murine fibroblasts as a feeder layer for keratinocytes. In this study, we report a simple procedure for growing human keratinocytes, isolated from adult skin, to generate an epidermal construct on a collagen layer alone. In this model, no human or murine feeder layers were used to amplify cell growth, and isolated keratinocytes were seeded directly at high cell density on the collagen-coated flasks or coverslips in an epithelial growth medium containing low calcium concentration. Morphological, immunochemical, and cytokinetic features of epithelial colonies grown on the collagen layer were typical of keratinocytes and were comparable with those reported for keratinocytes grown on a feeder layer. The stratification of keratinocytes generated 3-dimensional synthetic constructs displaying a tissue architecture comparable with that of natural epidermis. Epithelial cells expressed specific markers of keratinocyte terminal differentiation, including involucrin and filaggrin. Nevertheless, the number of cell layers was lower than in natural skin, and electron microscopical analysis revealed that the overall organization of these layers was poor compared with natural epidermis, including the formation of junctional complexes, basement membrane, and keratinization. The lack of epithelial-mesenchymal interactions that occur during skin histogenesis may account for such an incomplete maturation of epidermal constructs.     

Functional and phenotypic characterization of human keratinocytes expanded in microcarrier culture

Skin cells for transplantation are routinely prepared by growing patient keratinocytes in a semi-defined cocktail of growth factors, including serum and feeder cells. However, these reagents require substantial risk remediation and can contribute to transplant rejection. Microcarrier culture is an emerging technology that may allow the elimination of feeder cells whilst facilitating expansion of cultured keratinocytes. However, the behavior of keratinocytes in microcarrier culture and the potential of these cells to form an epidermis have been poorly defined. We characterized freshly isolated human keratinocytes cultured on CultiSpher-G microcarriers in the absence of murine feeder cells and assessed the potential of the keratinocytes to form an epidermis in an in vitro model. In a single passage, keratinocytes multiplied 44.9-fold in microcarrier-bioreactor culture in 17 days, whereas two-dimensional cultures reached confluence in 9 days and only expanded 7.4-fold. Histological characterization of keratinocytes on the microcarriers revealed that the cells were randomly distributed within these porous structures, however, not all pores contained cells. High-resolution microcomputed tomography imaging of the microcarriers confirmed limited interconnectivity of the pores. Immunoreactivity of specific epidermal markers was confirmed during cell expansion via immunohistochemistry. Despite the expression of differentiation markers, microcarrier-expanded keratinocytes retained the capacity to form an epidermis, as was evaluated using an in vitro human skin equivalent model. The epidermis formed by microcarrier-expanded keratinocytes in this model exhibited morphology similar to native skin. Significantly, the microcarrier technique successfully eliminates the need for a feeder cell layer and hence facilitates development of an improved culture system.     

The effect of self-designed bifunctional RGD-containing fusion protein on the behavior of human keratinocytes and dermal fibroblasts

In this study, self-designed bifunctional RGD-containing fusion protein (BFP) was grafted on the petri dish to evaluate its cytotoxicity and attachment efficiency on primary cultured keratinocytes and dermal fibroblasts. Two lengths of the GRGDS sequences were separately fused to the N-terminus and C-terminus of the Trichoderma koningii cellobiohydrolase I gene cellulose-binding domain, to serve as linking molecule between the cell and the substrate. The grafting procedure was no more labor-intensive and could be done just in aqueous condition itself. The epidermal keratinocytes and dermal fibroblasts, harvested and separated from human foreskin, were cultured in serum-free keratinocyte culture medium and DMEM, respectively. The BFP was dissolved in double-deionized water, and was prepared at different concentrations. The BFP solution was subsequently added into the petri dish for grafting. MTT assay, total DNA measurement, and lactate dehydrogenase analysis were used to evaluate the cell viability, cell proliferation, and cytotoxicity. The immunochemical stain and SEM examination were chosen to make sure that the cultured cells still kept in phenotype. The results showed that the self-designed BFP was successfully coated on the petri dish to improve the cells' adhesion. The whole coating procedure was just done in aqueous solution without any organic solvent being involved. This method was much simpler than the traditional one, and there was no possibility to damage the immobilized biomolecules. From the results of the study, BFP could enhance attachment of keratinocytes and dermal fibroblasts without losing normal cell morphology and keep keratinocytes on the desired differentiation pathway. We believe that coating BFP on petri dish not only enhanced the keratinocyte attachment but also promoted keratinocytes proliferation. We suggest that the self-designed BFP has a great potential to apply on surface modification for the tissue-engineering scaffolds in the future.     

Growth factor responsiveness declines during adulthood for human skin-derived cells

Keratinocytes and fibroblasts were obtained from upper arm biopsies of young (22-27 years) and old (60-82 years) adult donors. Keratinocytes were grown in serum-free medium containing variable quantities of either epidermal growth factor (EGF) or a bovine hypothalamic extract known to contain keratinocyte growth factor (KGF). Fibroblasts were grown in serum-free medium containing variable quantities of EGF or insulin. Paired keratinocyte cultures were plated in serum-free medium containing 20% newborn keratinocyte-conditioned medium (NM) or 20% control conditioned medium (CM). Newborn foreskin keratinocytes were plated in 20% conditioned media derived from newborn, young adult or old adult keratinocyte cultures. In spite of large inter-donor variability, keratinocyte growth significantly decreased with age (0.05 greater than P greater than 0.01). Cell yield at 7 days showed an 8-fold increase for young adults over the KGF dose range treated, but only a 4-fold increase for old adults. Young adult cells in varying concentrations of EGF achieved 3-fold to 5-fold higher densities than old, although EGF was not stimulatory for either adult age group. Donor age-associated loss of growth factor responsiveness was confirmed with dermal fibroblasts derived from the same biopsies. Newborn but not adult keratinocytes were stimulated by NM, while newborn cells were not stimulated by either young or old adult conditioned media (YM or OM). An epidermal proliferation index, incorporating both donor cell yield and cell yield of newborn cells in donor conditioned medium, was significantly different (P less than 0.01) for newborn vs. young or old adult cells. Our findings confirm that a decreased proliferative capacity is measurable within adulthood, and suggest that this decrease may be due to a reduced ability to synthesize or respond to mitogens, including autocrine factors.     

Human galectins induce conversion of dermal fibroblasts into myofibroblasts and production of extracellular matrix: potential application in tissue engineering and wound repair

Members of the galectin family of endogenous lectins are potent adhesion/growth-regulatory effectors. Their multifunctionality opens possibilities for their use in bioapplications. We studied whether human galectins induce the conversion of human dermal fibroblasts into myofibroblasts (MFBs) and the production of a bioactive extracellular matrix scaffold is suitable for cell culture. Testing a panel of galectins of all three subgroups, including natural and engineered variants, we detected activity for the proto-type galectin-1 and galectin-7, the chimera-type galectin-3 and the tandem-repeat-type galectin-4. The activity of galectin-1 required the integrity of the carbohydrate recognition domain. It was independent of the presence of TGF-β1, but it yielded an additive effect. The resulting MFBs, relevant, for example, for tumor progression, generated a matrix scaffold rich in fibronectin and galectin-1 that supported keratinocyte culture without feeder cells. Of note, keratinocytes cultured on this substratum presented a stem-like cell phenotype with small size and keratin-19 expression. In vivo in rats, galectin-1 had a positive effect on skin wound closure 21 days after surgery. In conclusion, we describe the differential potential of certain human galectins to induce the conversion of dermal fibroblasts into MFBs and the generation of a bioactive cell culture substratum.     

In situ image analysis of interactions between normal human keratinocytes and fibroblasts cultured in three-dimensional fibrin gels

The non-invasive investigation of different cells to interact and become spatially organised in a three-dimensional (3D) environment or scaffold is an important challenge in tissue engineering and tissue physiology. The aim of the present study was to develop 3D cell culture systems using fibrin gels, which would allow for the single and co-culture of different cell types with in situ image analysis. Two chambers were constructed for mono-culture and co-culture of human dermal fibroblasts and keratinocytes. During cell culture, in situ imaging and morphological characterisation of cells was assessed using brightfield light and/or fluorescence microscopy, and later confirmed by staining of fixed cells using immunofluorescence microscopy. The results showed that it was possible to investigate fibroblast and keratinocyte interactions in a fibrin scaffold for at least 12 days. Using this model system it was found that when a co-culture of fibroblasts and keratinocytes were plated on top of the fibrin gels, fibroblasts were seen to migrate into the gels within 2-3 days in contrast to keratinocytes, which did not enter. However, keratinocytes were found to retard fibroblast migration into gels when compared to fibroblasts cultured on their own, illustrating the dependency of intracellular communication on cell position for reconstructive approaches.     

Membranes from acrylonitrile-based polymers for selective cultivation of human keratinocytes

A cell carrier made from synthetic material supporting selective growth of keratinocytes is a promising approach to avoid the phenomenon of fibroblast overgrowth during in vitro culture of skin substitutes. Therefore, we investigated polymer membranes made of polyacrylonitrile and copolymers of acrylonitrile and N-vinylpyrrolidone (NVP) for their ability to support selectively the growth of keratinocytes. It was found that a copolymer with an NVP-content of 30% (NVP30) supports growth of human keratinocyte cell line (HaCaT) cells and inhibits fibroblast growth under serum-containing conditions. Cell proliferation of HaCaT cells was measured over 14 days. If both cell types were cultured under serum-free conditions for initial adhesion over 6 h on these NVP30 polymers, they adhered to the same extent. Long-term experiments over 7 days were performed as a coculture of both cell types showing that HaCaT cells had a growth advantage that seems to be related to the paracrine activity of contaminating fibroblasts. As a result, confluent layers of HaCaT cells were obtained with small numbers of remaining fibroblasts. The new poly [acrylonitrile-co(NVP) membranes seem to be a promising culture system for the production of epidermal transplants.     

Recapitulation of oral mucosal tissues in long-term organotypic culture

To test the influence of fibroblasts on epithelial morphology and expression of keratinocyte proteins and barrier lipids, we bioengineered homotypic and heterotypic oral mucosae and skin using cultured adult human cells. Fibroblasts were allowed to modify collagen type I gels for 2 weeks before keratinocytes were added. The organotypic cultures were then grown at the air-liquid interface for 4 weeks. In homotypic combinations, epithelial morphology and protein expression closely mimicked those in vivo. In heterotypic combinations, the morphology resembled that in vivo and keratinocytes expressed their typical markers, except when skin keratinocytes were recombined with alveolar fibroblasts; they expressed K19, K4, and K13, which is similar to oral mucosal epithelia rather than to the epidermis. Morphologically, the stratum corneum layers were typical for the epithelial tissues. Grafting the bioengineered cultures to the backs of Nude mice did not change the results, suggesting that our findings are not merely a culture phenomenon. Lipid profiles of the homotypic combinations mimicked the profiles found in the normal epithelial tissues, except that the engineered alveolar epithelium expressed more ceramide 2 than that in vivo. In the heterotypic combinations, keratinocytes appeared to control the lipid profile, except in the combination of skin keratinocytes with alveolar fibroblasts, wherein the ceramide profile appeared to be partly that of alveolar epithelium and partly that of epidermis. These results suggest that cultured adult fibroblasts and keratinocytes are sufficient to recapitulate graftable oral tissues, and, except for alveolar fibroblasts, the type of fibroblast had little influence on keratinocyte differentiation.     

A denatured collagen microfiber scaffold seeded with human fibroblasts and keratinocytes for skin grafting

Biomaterial scaffolds are categorized into artificial or natural polymers, or combinations of the two. Artificial polymers often undergo serum protein adsorption, elicit foreign body and encapsulation immune responses post-implantation. Large pore bovine electrospun collagen I was therefore screened as a candidate for human keratinocyte and fibroblast cell scaffolds. Human HaCaT keratinocyte and dermal fibroblasts were seeded on electrospun denatured collagen I microfiber (DCM) scaffolds and after 72 h Livedead(?) assays performed to determine adhesive cell, survival and scaffold penetration. Both keratinocytes and fibroblasts attached to and survived on DCM scaffolds, however only fibroblasts migrated over and into this biomaterial. HaCaT keratinocytes remained largely stationary on the scaffold surface in discrete islands of monolayered cells. For this reason, normal human epidermal keratinocyte (NHEK) scaffold interactions were assessed using scanning and transmission electron microscopy (EM) that demonstrated DCM scaffolds comprised networks of interlocking and protruding collagen fibers with a mean diameter of 2-5 μm, with a mean inter-fiber pore size of 6.7 μm (range 3-10 μm) and scaffold thickness 50-70 μm. After 72 h the keratinocytes and fibroblasts on DCM scaffolds had attached, flattened and spread over the entire scaffold with assembly of lamellapodia and focal adhesion (FA)-like junctions. Using transmission EM, NHEKs and HaCaT keratinocytes assembled desmosomes, lamellapodia and FA junctions, however, neither hemidesmosomes nor basal lamina were present. In long term (21 day) co-culture fibroblasts migrated throughout the scaffold and primary keratinocytes (and to a lesser extend HaCaTs) stratified on the scaffold surface forming a human skin equivalent (HSE). In vivo testing of these HSEs on immunocompetent (BalbC) and immunodeficient (SCID) excisionally wounded model mice demonstrated scaffold wound biocompatibility and ability to deliver human cells after scaffold biodegradation.     

Influence of an epidermal cell extract on skin healing and scar formation

We have examined the possible regulatory role of epidermal cell extract(s) (ECE) on skin cells, namely fibroblasts and keratinocytes, both in vivo and in vitro with particular reference to modification of scar formation. In an experimental wound model in pigs, it was found that extracts of cultured human and pig keratinocytes stimulated replication of epidermal cells and their migration from wound edges and remnants of hair follicles and sebaceous glands, together with hair growth, but at the same time suppressed fibroblast proliferation in the dermis. Sections of healing skin wounds that had been treated with ECE showed the presence of a thick layer of epidermal cells lying on relatively sparse dermis. There was little or no contraction in treated wounds and scarring was minimal. Clinical studies of granulomatous lesions of horses and ulcerated wounds in dogs that had been treated with ECE supported these findings. In contrast to its apparently general stimulation of keratinocytes in vivo, ECE had a highly selective effect in vitro on epidermal cells plated at low density in the absence of a feeder layer, which suggests that its action in vivo may be confined to a specific sub-population of rapidly proliferating keratinocytes or alternatively mediated through a second messenger from another type of cell. The inhibitory effect of epidermal cell extract on fibroblasts in vitro was shown by its ability to prevent the contraction of collagen sponges by fibroblasts. These results suggest an important role for epidermal factors in the growth regulation of both epidermal and dermal cells during wound healing.     

In vitro cytotoxicity testing of a nanocrystalline silver dressing (Acticoat) on cultured keratinocytes

Acticoat is a polyethylene mesh coated with nanocrystalline silver. It has been used widely as a dressing for chronic wounds, acute partial-thickness burn wounds and donor sites. In this study, the in vitro cytotoxicity of Acticoat on cultured keratinocytes is tested. Human keratinocytes are cultivated on a pliable hyaluronate-derived membrane (Laserskin) using dermal fibroblasts as the feeder layer. When the cultured Laserskin (CLS) is subconfluent it is covered by Acticoat, which is exposed to water (Group 1), phosphate-buffered saline (Group 2) or culture medium (Group 3). The control group is not exposed to the Acticoat. After 30 minutes incubation at 37 degrees C, the inhibitory effect of the nanocrystalline silver on keratinocyte growth is measured by an MTT assay. Compared with the control, the relative viability of the CLS dropped to 0%, 0% and 9.3%, respectively. Thus, Acticoat is cytotoxic to cultured keratinocytes and should not be applied as a topical dressing on cultured skin grafts.     

Structure of a collagen-GAG dermal skin substitute optimized for cultured human epidermal keratinocytes

Collagen and glycosaminoglycan (GAG) dermal skin substitutes (membranes) were studied as substrates for cultured human epidermal keratinocytes. Structure of dermal substitutes was optimized for pore size to promote ingrowth of fibrovascular tissue from the wound bed and for culture of human keratinocytes of the membrane's surface. Pore size of the freeze-dried material was regulated by control of the temperature of freezing between -50 degrees C and -20 degrees C and by concentration of starting materials between 0.17% and 1.62% wt/vol. A nonporous surface of collagen-GAG was laminated to the membranes to provide a planar substrate for cultured epidermal keratinocytes. Thickness of dermal substitutes was regulated by control of the volume and concentration of starting materials. Biotin was conjugated to solubilized collagen for binding with avidin of specific quantities of biologically active molecules. The optimized membranes are suitable substrates for the culture of human epidermal keratinocytes, and together with the cells yield a composite material that is histologically similar to skin.     

Human skin cell cultures onto PLA50 (PDLLA) bioresorbable polymers: influence of chemical and morphological surface modifications

Poly(alpha-hydroxy acid)s derived from lactic and glycolic acid are bioresorbable polymers which can cover a large range of thermal, physical, mechanical, and biological properties. Human keratinocytes have been shown as able to grow on a poly(DL-lactic acid) film. However the keratinocyte growth was delayed with respect to culture on standard tissue culture polystyrene, even though the same plateau level was observed after 2 weeks. In order to improve the performance of poly(DL-lactic acid) films as skin culture support, their surface was modified by creating tiny cavities using a method based on the leaching out of poly(ethylene oxide) from poly(lactic acid)-poly(ethylene oxide) heterogeneous blends. The surface of the films was also chemically modified by alkaline attack with sodium hydroxide and by type-I collagen coating. Murine fibroblast cell line and primary cultures of human fibroblasts and of two types of keratinocytes were allowed to adhere and to grow comparatively on the different films. The presence of cavities affected neither the adhesion of dermal fibroblasts nor that of keratinocytes. Only keratinocyte proliferation was significantly reduced by the presence of cavities. Collagen coating improved skin cell adhesion and proliferation as well, except in the case of murine fibroblasts. In the case of the NaOH treatments, similar trends were observed but their extent depended on the treatment time. In the case of chemical modifications, fluorescence microscopy bore out adhesion and proliferation tendencies deduced from MTT tests.     

包皮成纤维细胞作为饲养层对人胚胎生殖细胞生长的影响

目的 以人包皮成纤维细胞为饲养层体外培养人胚胎生殖细胞,观察饲养层对其生长的影响,并对人胚胎生殖细胞进行鉴定。 方法 分离培养4～5岁小儿包皮成纤维细胞,取P₃～P₃₀代细胞用丝裂霉素灭活后铺板备用,酶联免疫吸附测定（ELISA）双抗夹心法检测其分泌物成纤维细胞生长因子（FGF）的含量;分离培养5～11周人胚胎原始生殖细胞,在不添加任何细胞因子的人包皮成纤维细胞饲养层上培养人胚胎生殖细胞;细胞化学法检测人胚胎生殖细胞碱性磷酸酶的活性,免疫细胞化学法检测其胚胎表面特异性抗原SSEA-1及SSEA-4的表达,RT-PCR法检测Oct-4的表达。 结果 包皮成纤维细胞可以传60代以上,P₃～P₃₀代均适宜作饲养层;P₃～P₃₀代包皮成纤维细胞上清液中FGF含量在(172.09±2.66)pg/L～(245.25±1.66)pg/L之间波动。分离培养的人胚胎生殖细胞在饲养层上可形成典型的胚胎生殖细胞集落,体外连续培养超过3代,其碱性磷酸酶活性呈强阳性,集落未分化标志检测显示SSEA-l、SSEA-4呈阳性,Oct-4表达阳性。 结论 用人包皮成纤维细胞作为饲养层能支持人胚胎生殖细胞的生长并维持未分化状态。     

Development of defined media for the serum-free expansion of primary keratinocytes and human embryonic stem cells

Primary keratinocyte (Kc) cells and human embryonic stem (hES) cells are routinely propagated on a mouse fibroblast feeder layer in media containing fetal bovine serum or other nondefined factors. One disadvantage of using these nondefined factors is that they may inadvertently contaminate the culture system with infectious agents; thus, there remains a need to develop safe culture conditions free from poorly defined and/or animal products. Our laboratory has discovered that growth factors (GFs) and vitronectin (VN) can bind to each other resulting in synergistic short-term functional effects in several cell types. The aim of the current study was to determine whether primary Kc and hES cells can be established and serially propagated serum-free using medium containing VN, insulin-like growth factor-I, and insulin-like growth factor binding protein-3 (VN:GF). Here we demonstrate that primary Kc cells can be isolated, established, serially propagated, and re-form an epidermal layer using the VN:GF combination. Additionally, cell proliferation studies indicate that the Kcs proliferate using the VN:GF combination at a rate comparable to cells grown using serum. Similarly, we verified that this VN:GF combination could be employed for the serial propagation of hES cells. Importantly, both the Kc and hES cells retain their undifferentiated phenotype when cultured using the VN:GF combinations as a serum-free medium for up to 4 passages for Kc and at least 10 passages for hES cells as indicated by the expression of a range of cell surface markers. This study demonstrates that the novel, fully defined VN:GF medium is a viable alternative to media containing serum and highlights the potential of this technology for generating therapeutically viable cells and tissues.     

Oral fibroblasts produce more HGF and KGF than skin fibroblasts in response to co-culture with keratinocytes

The production of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) in subepithelial fibroblasts from buccal mucosa, periodontal ligament, and skin was determined after co-culture with keratinocytes. The purpose was to detect differences between the fibroblast subpopulations that could explain regional variation in epithelial growth and wound healing. Normal human fibroblasts were cultured on polystyrene or maintained in collagen matrix and stimulated with keratinocytes cultured on membranes. The amount of HGF and KGF protein in the culture medium was determined every 24 h for 5 days by ELISA. When cultured on polystyrene, the constitutive level of KGF and HGF in periodontal fibroblasts was higher than the level in buccal and skin fibroblasts. In the presence of keratinocytes, all three types of fibroblasts in general increased their HGF and KGF production 2-3 times. When cells were maintained in collagen, the level of HGF and KGF was decreased mainly in skin cultures. However, in oral fibroblasts, induction after stimulation was at a similar level in collagen compared to on polystyrene. Skin fibroblasts maintained in collagen produced almost no HGF whether with or without stimulation. The results demonstrate that the secretion of KGF and HGF in both unstimulated fibroblasts and in fibroblasts co-cultured with keratinocytes is dependent on the type of fibroblasts. In general, the periodontal fibroblasts had the highest level of cytokine production. This high level of growth factor production may influence the proliferation and the migration of junctional epithelium and thereby influence the development of periodontal disease.