Culture of Keratinocytes for Transplantation without the Need of Feeder Layer Cells

Coolen, Neeltje A.; Verkerk, Michelle; Reijnen, Linda; Vlig, Marcel; Bogaerdt, Antoon J. van den; Breetveld, Melanie; Gibbs, Susan; Middelkoop, Esther; Ulrich, Magda M. W.

doi:10.3727/000000007783465046

Cited by 54 publications

(48 citation statements)

References 44 publications

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“…Although Coolen et al (29) showed that keratinocytes can be cultured without a fibroblast feeder layer and fetal calf serum, the present results demonstrated that in the modified medium and onto a type I collagen scaffold (as a feeder layer), keratinocyte cell growth was greater than the condition without a feeder layer, and this is consistent with certain previous studies by Gingras et al (31) and Arpornmaeklong et al (32) in 2007. However, a long-term follow-up study is required to more precisely evaluate its fate following proper grafting.…”

Section: Discussionsupporting

confidence: 82%

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Comparison of the enzymatic and explant methods for the culture of keratinocytes isolated from human foreskin

et al. 2015

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Abstract. Currently, culture and growth keratinocytes are important stages in achieving a reliable and reproducible skin tissue. In the present study, two different methods, enzymatic and explant methods, for keratinocytes isolation from human foreskin were compared. Foreskins were cut into 2-3 mm pieces and placed in trypsin at 4˚C overnight for separation of the epidermis from the dermis. Subsequently, these samples were divided into two groups: i) Keratinocytes separated from the epidermis by trypsin and ii) by the explant method. These keratinocytes were divided into two groups: i) With no feeder layer and ii) onto a type I collagen scaffold. The cells were evaluated using immunocytochemistry and 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI) staining. In the enzymatic treatment, after 7-10 days no attached cells were found in the cell culture dishes. In the explant method, keratinocytes were separated after ~24 h, attached rapidly and formed big colonies into a collagen scaffold. In the absence of a feeder layer, small colonies were developed with rapid loss of proliferation within 2-3 days. Keratinocytes showed positive immunoreactivity for the pan-cytokeratin marker and keratinocytes' nuclei were clearly observed. This method could be applied and developed as a component of skin substitutes to treat burns and wounds and also in laboratory testing. IntroductionThe skin is the largest organ in the body that is divided into two anatomically distinct regions, the dermis and epidermis. The normal structure and function of this organ is dependent on the intact epidermis anchored to its vascular, elastic dermis (1,2). Fibroblasts are the most prevalent cell types in the dermis, which produce different growth factors that induce proliferation of keratinocytes in vivo and in vitro (3). The principal cell type of the epidermis is the keratinocyte (1,4), which is a small epithelial cell, located at the top of the epidermal basal membrane and characterized by a low division rate (5,6).Thus far, various enzymatic methods for dermal-epidermal separation have been applied (6-8). For instance, trypsin separates suprabasal hemidesmosomes that causes the basal layer cells to attach to the dermal layer (6). Thermolysin is another enzyme that selectively separates desmosomes (5,9) and is able to separate the epidermis at the basal membrane zone level (5,10).Although the dissociation method of keratinocytes in primary culture is well-established, attempts to acquire purified adult stem-cell like⁄progenitor keratinocytes from whole human skin are still ongoing. In particular, different techniques are currently being applied to achieve high purity or homogeneous primary cultures enriched in keratinocyte progenitor⁄precursor cells. These include filtration, density gradient centrifugation and fluorescence-activated cell sorting using cell surface antibodies, as well as differential adhesion to enrich the cells that rapidly attach to particular substrates (11).A previous study showed that when keratinocytes/precursor cells...

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Section: Discussionsupporting

confidence: 82%

“…There are numerous studies that have focused on the development of nutritionally optimized, readily defined, reproducible media and culture conditions for keratinocyte cells (26)(27)(28)(29). The aims of the present study were not to establish a new medium, and therefore, the Rheinwald and Green (30) protocol was applied.…”

Section: Discussionmentioning

confidence: 99%

Comparison of the enzymatic and explant methods for the culture of keratinocytes isolated from human foreskin

et al. 2015

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“…Representative overlay histograms showed no difference in the positive staining for (a) the proliferation marker cytokeratin 14 (K14) and (c) the wound healing marker cytokeratin 6 (K6) between AK in different culture conditions. Anova all pair-wise comparison showed no statistically significant differences in the percentage of positive events of (b) K14 and (d) K6 between the different culture conditions (n = 3, p = 0.05) towards developing and testing defined medium conditions (Boyce and Ham 1983;Sun et al 2004;Coolen et al 2007;Mujaj et al 2010;De Corte et al 2011). In these experiments we did not test defined medium conditions beyond the use of EpiLife with EDGS.…”

Section: Discussionmentioning

confidence: 99%

A fully autologous co-culture system utilising non-irradiated autologous fibroblasts to support the expansion of human keratinocytes for clinical use

et al. 2011

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Autologous keratinocytes can be used to augment cutaneous repair, such as in the treatment of severe burns and recalcitrant ulcers. Such cells can be delivered to the wound bed either as a confluent sheet of cells or in single-cell suspension. The standard method for expanding primary human keratinocytes in culture uses lethally irradiated mouse 3T3 fibroblasts as feeder cells to support keratinocyte attachment and growth. In an effort to eliminate xenobiotic cells from clinical culture protocols where keratinocytes are applied to patients, we investigated whether human autologous primary fibroblasts could be used to expand keratinocytes in culture. At a defined ratio of a 6:1 excess of keratinocytes to fibroblasts, this co-culture method displayed a population doubling rate comparable to culture with lethally irradiated 3T3 cells. Furthermore, morphological and molecular analysis showed that human keratinocytes expanded in co-culture with autologous human fibroblasts were positive for proliferation markers and negative for differentiation markers. Keratinocytes expanded by this method thus retain their proliferative phenotype, an important feature in enhancing rapid wound closure. We suggest that this novel co-culture method is therefore suitable for clinical use as it dispenses with the need for lethally irradiated 3T3 cells in the rapid expansion of autologous human keratinocytes.

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“…The major issue with this study was the use of the irradiated mouse 3T3 feeder layer cells, which were regarded as a potential risk due to the possible transfer of disease, such as animal viruses or prions. In addition, the low number of CIN keratinocytes rescued was frequently associated with contaminant human fibroblasts, which rapidly overgrew the epithelial cells in culture (11)(12)(13). Additionally, the growth of the keratinocytes was constrained due to the limited space during the course of culture.…”

Section: Introductionmentioning

confidence: 99%

A modified method for the culture of naturally HPV-infected high-grade cervical intraepithelial neoplasia keratinocytes from human neoplastic cervical biopsies

2016

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Abstract. Few studies on cervical intraepithelial neoplasia (CIN) keratinocyte cultures are available due to the numerous technical and methodological problems associated with the in vitro cultivation of these cells. The present study investigated an applicable and effective method for the in vitro cultivation of high-grade CIN keratinocytes from human neoplastic cervical biopsies. Human neoplastic cervical tissue sections were obtained and digested using type I collagen in order to dissociate the cells. The cells were seeded in tissue culture plastic plates that were coated with rat tail collagen type I and contained modified keratinocyte serum-free medium (K-SFM) supplemented with 5% fetal bovine serum. The medium was replaced with K-SFM on days 3, 5 and 7, respectively. The unattached cells were recovered and the cell viability was determined accurately using the Trypan Blue exclusion method. The expression of keratin 14 (K14), keratin 19 (K19), keratin 17 (K17) and P63 was assayed using immunofluorescence in order to identify the presence of CIN keratinocytes. The present results indicated that the attachment rate of CIN keratinocytes significantly increased between 56.75±1.76% on day 3 and 77.09±3.55% on day 5, and became relatively stable between days 5 and 7. The cell viability significantly decreased between 83.00±0.50% on day 5 and 68.17±1.04% on day 7. The passaged CIN keratinocytes maintained the original unequally sized, abnormally shaped morphology and did not undergo differentiation. In addition, the passaged CIN keratinocytes exhibited the same human papilloma virus (HPV) genotype that was detected in the original primary cells. K14 and K19 were expressed in the majority of the normal and CIN keratinocytes, whereas K17 and P63 were expressed only in high-grade CIN keratinocytes. The present study proposes a simple and practical method for rapidly obtaining highly purified naturally HPV-infected high-grade CIN keratinocytes from small neoplastic cervical tissues, and provides an appropriate first medium change time for the primary culture of CIN keratinocytes. IntroductionCervical cancer is the most common gynecological cancer worldwide and is usually preceded by a long phase of pre-malignant disease (1,2). These pre-malignant changes represent a spectrum of histological abnormalities ranging between cervical intraepithelial neoplasia (CIN) I (mild dysplasia), CIN II (moderate dysplasia) and CIN III (severe dysplasia/carcinoma in situ) (3,4). In the majority of patients, CIN I will regress spontaneously and is not usually precancerous; however, 20-45% of untreated CIN II and III lesions will persist and progress to cervical cancer (5,6). Therefore, CIN II and CIN III are considered to be high-grade precancerous lesions. Previous studies have established that the majority of high-grade CIN cases are caused by persistent oncogenic human papilloma virus (HPV) infection, and a long latency period of viral infection is required for CIN progression (7-9). However, viruses do not metabolize, and require a...

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Culture of Keratinocytes for Transplantation without the Need of Feeder Layer Cells

Cited by 54 publications

References 44 publications

Comparison of the enzymatic and explant methods for the culture of keratinocytes isolated from human foreskin

Comparison of the enzymatic and explant methods for the culture of keratinocytes isolated from human foreskin

A fully autologous co-culture system utilising non-irradiated autologous fibroblasts to support the expansion of human keratinocytes for clinical use

A modified method for the culture of naturally HPV-infected high-grade cervical intraepithelial neoplasia keratinocytes from human neoplastic cervical biopsies

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