8073 - ISSN 2307 Culture technique of rabbit primary epidermal keratinocytes (original) (raw)
Related papers
Serum-Free Serial Culture of Adult Human Keratinocytes From Suction-Blister Roof Epidermis
Journal of Investigative Dermatology, 1987
Coatin g cell culture fl as ks with natural extracellul ar matrix (ECM) en hanced the culture of adult hum an keratinocytes from suction-blister roof epidermis in an en vironment without fetal ca lf serum (FCS), bo vin e pituitary ex tracts or cellul ar feeder layers. A hi g her in ciden ce of cell attachm ent on natural ECM was observed than 011 coll agen and human fibro nectins(HFN)-coated plas tic di shes , and natural ECM was necessa ry for g rowth and proliferation of attached cells under th e culture condition s used. Cells in T he suction-blister m eth od is convenient and suitable to ob tain the epidermis from in fa nts and adu lts beca use it ca uses no pain and it produces no scar on the exa mined subjects. The ro ofs of the suction blisters are reported to be composed of pure epiderma l cells lack in g an y derm al cells [1]. Although there have been many tec hnica l ap proaches to the culture of hum an keratinocytes, m ost culture m ethods need a cellular feeder la yer and /or se rum. The presence of serum or other cell types, ho wever, prohibits the interpretation of certain types or-experiments on cellular attachm ent or of protein synthes is. and m ay chan ge the morphology and perh aps the biology of the cultured cells [2]. Recently. m eth od s for serum-free culture of hum an keratinocytes have been reported [2-5]. in most of w hi ch coll agen and other matrix-coated gro wth substrata are used. or in w hi ch pituitary extracts (PE) replace fetal calf se rum (FCS). Accumulatin g evidence o n the bio lo gic fe atures of culfured keratinocytes in serum-free env iron m ents has provided new insig hts in bas ic in ves ti ga ti ve dermatology and so m e striking clinical ap pli ca tions. It has been a lo ng-range goal of many investigators to isolate and to grow human epidermal keratinocy tes from both norm al and diseased hum an skin in suffi cient quantities for va ri o us biological studies [6]. This has not been feasible to date beca use Manuscript
Experimental model of cultured keratinocytes
Acta Cirurgica Brasileira, 2003
The bioengineering research is essential in the development of ideal combination of biomaterials and cultured cells to produce the permanent wound coverage. The experimental model of cultured keratinocytes presents all steps of the culture, since the isolation of the keratinocytes, preparation of the human acellular dermis, preparation of the composite skin graft and their elevation to the air-liquid interface. The research in cultured keratinocytes model advances in two main ways: 1. optimization of the methods in vitro to the skin cells culture and proliferation and 2. developing biomaterials that present similar skin properties.
The Keio Journal of Medicine, 1990
An optimum culture condition was established for our keratinocyte cell culture system from human adult skin using 3T3 feeder cells. Calcium ion (Ca++) concentration was found to be critical and cells grew best at the Ca++ concentration of 0.2mM. Keratinocyte proliferation was promoted when 0.4ƒÊg/ml hydrocortisone and 7ƒÊg/ml insulin were added. However, epidermal growth factor, cholera toxin and transferrin did not show stimulating effects on multiplication of human keratinocytes in our culture system. The epidermal sheets grown in vitro under this optimized condition were transplanted onto athymic mice, and a short term of take was observed.
Journal of Cellular Biotechnology, 2017
Keratinocytes are the main cell population in the epidermis, where they coexist with a variety of other cell types. Their successful isolation and cultivation have afforded opportunities to study epidermal functions. Human keratinocytes have been studied most extensively, but their source is limited by the skin supply. In a previous work, we developed an in vitro co-culture model of porcine keratinocytes with porcine sensory neurites to investigate functional interaction. However, a detailed description of the isolation of porcine keratinocytes and their culture conditions has not been given in detail. Here, we present the isolation procedure and a characterization of keratinocytes derived from newborn piglets, using simple assays based on conventional and fluorescence microscopy. Media, coating substrates and plating densities were tested with respect to cell viability, proliferation and morphology. Growing keratinocytes in EpiLife keratinocyte growth medium (EKGM) on human collagen type I substrate was best to support proliferation. The minimum plated density was 500 viable cells/cm 2 for primary and 1000 viable cells/cm 2 for subcultured cells. Population doubling (PD) and generation time (tg) depended on the plating densities. Keratinocytes seeded at a density of 5000 viable cells/cm 2 had a PD of 4.36 ± 0.60 per passage and tg of 1.69 ± 0.24 days. Our results show that the optimal isolation and culture conditions for keratinocytes from piglets differ from those for keratinocytes from adult pigs and humans. Thus, the information obtained from the characterization allowed the performance and optimization of a co-culture and contributes to further investigations in epidermal homeostasis and cutaneous sensation.
Culture of Keratinocytes for Transplantation Without the Need of Feeder Layer Cells
Cell Transplantation, 2007
Patients with large burn wounds have a limited amount of healthy donor skin. An alternative for the autologous skin graft is transplantation with autologous keratinocytes. Conventionally, the keratinocytes are cultured with mouse feeder layer cells in medium containing fetal calf serum (FCS) to obtain sufficient numbers of cells. These xenobiotic materials can be a potential risk for the patient. The aim of the present study was to investigate if keratinocytes could be expanded in culture without the need of a feeder layer and FCS. Keratinocytes were cultured on tissue culture plastic with or without collagen type IV coating in medium containing Ultroser G (serum substitute) and keratinocyte growth factor (KGF). An in vitro skin equivalent model was used to examine the capacity of these cells to form an epidermis. Keratinocytes in different passages (P2, P4, and P6) and freshly isolated cells were studied. Keratinocytes grown on collagen type IV were able to form an epidermis at higher passage numbers than cells grown in the absence of collagen type IV (P4 and P2, respectively). In both cases the reconstructed epidermis showed an increased expression of Ki-67, SKALP, involucrin, and keratin 17 compared to normal skin. Only 50,000 keratinocytes grown on collagen type IV in P4 were needed to form 1 cm 2 epidermis, whereas 150,000 of freshly isolated keratinocytes were necessary. Using this culture technique sufficient numbers of keratinocytes, isolated from 1 cm 2 skin, were obtained to cover 400 cm 2 of wound surface in 2 weeks. The results show that keratinocytes can be cultured without the need of a fibroblast feeder layer and FCS and that these cells are still able to create a fully differentiated epidermis. This culture technique can be a valuable tool for the treatment of burn wounds and further development of tissue engineered skin.
A Comprehensive Comparative Characterization of Keratinocytes; The Significant Wound Healing Agents
Keratinocytes are the main components of skin epidermis constituting more than 90% of it which are responsible to regulate skin regeneration during external or internal injury. These cells can be found in a heterogenic form containing proliferative, terminally differentiated and transit amplifying (TA) cells located in basal layer, outer keratinized layer and intermediate layers, respectively when isolated from an adult skin. Efforts are going on to characterize keratinocytes precisely as in comparison with widely used stem cells, mesenchymal stem cells (MSCs) and their application in cutaneous wound healing as a translational approach in regenerative medicine. In this work, we have applied a comprehensive approach to identify and characterize keratinocytes which are a valuable tool in wound healing. Keratinocytes have successfully been isolated from adult skin and the ratio of proliferative, terminally differentiated and TA cells based on the expression of α6-integrin and CD71 was studied using flow cytometry after 7, 14 and 21 days of their culture in vitro. RT and qRT-PCR was applied to study the change in genetic expression and relativity of cytokeratin markers with the passage of time. PopUp culturing and population doubling time was performed to study the proliferative potential of heterogenic population of cells. Their colonogenicity and wound healing potential were also studied to explain their healing behaviour during the time of injuries. 47% keratinocytes after 7 days, 62% after 14days and 93% keratinocytes after 21 days of culturing. Expression of CD71 was also observed in cells as, 5% after 7 days, 22% after 14 days and 66% of keratinocytes after 21 days were positive for CD71 expression. Gradual increased expression of genetic markers, K10, K14, INV and P63 was observed using qRT-PCR. Studying comparative data, it can be concluded that keratinocytes after 14 days of culturing are better to use for clinical purposes. This comprehensive protocol could be a valuable addition in cutaneous biology helping researchers to identify keratinocytes and to use them for their pre-clinical studies.
The Journal of Cell Biology, 1978
A population of neonatal mouse keratinocytes (epidermal basal cells) was obtained by gentle, short-term trypsin separation of the epidermal and dermal skin compartments and discontinuous Ficoll gradient purification of the resulting epidermal cells. Over 4--6 wk of culture growth at 32--33 degrees C, the primary cultures formed a complete monolayer that exhibited entire culture stratification and upper cell layer shedding. Transmission and scanning electron microscopy demonstrated that the keratinocyte cultures progressed from one to two cell layers through a series of stratification and specialization phenomena to a six to eight cell layer culture containing structures characteristic of epidermal cells and resembling in vivo epidermal development. The temporal development of primary epidermal cell culture specialization was confirmed by use of two histological techniques which differentially stain the specializing upper cell layers of neonatal mouse skin. No detectable dermal fibroblast co-cultivation was demonstrated by use of the leucine aminopeptidase histochemical technique and routine electron microscope surveillance of the cultures. Incorporation of [3H]thymidine ([3H]Tdr) was greater than 85% into DNA and was inhibited by both 20 micron cytosine arabinoside (Ara-C) and low temperature. Autoradiography and 90% inhibition of [3H]Tdr incorporation by 2 mM hydroxyurea indicated that keratinocyte culture DNA synthesis was scheduled (not a repair phenomenon). The primary keratinocytes showed an oscillating pattern of [3H]Tdr incorporation into DNA over the initial 23--25 days of growth. Autoradiography demonstrated that the cultures contained 10--30% proliferative stem cells from days 2-25 of culture. The reproducibility of both the proliferation and specialization patterns of the described primary epidermal cell culture system indicates that these cultures are a useful tool for investigations of functioning epidermal cell homeostatic control mechanisms.
International Journal of Molecular Sciences
For the development of advanced therapies, the use of primary cells instead of cell lines is preferred. The manufacture of human tissue-engineered skin substitutes requires efficient isolation and culture protocols allowing a massive expansion of the cells in culture from an initial specimen of a minimal size. This study compared two skin cell isolation protocols, routinely applied in two clinical laboratories. Epithelial (keratinocytes) and dermal (fibroblasts) cells were isolated and cultured from three human skin biopsies (N = 3). The two-step digestion protocol (LOEX-Protocol) firstly used thermolysin to enzymatically disrupt the dermal–epidermal junction while, for the one-step digestion protocol (UPCIT-Protocol), mechanical detachment with scissors was applied. Then, the epidermal and dermal layers were digested, respectively, to achieve cell isolation. The cell size, viability, yield and growth were analyzed over five passages (P). The colony-forming efficiency (CFE) and Kera...
An improved method for culture of epidermal keratinocytes from newborn mouse skin
Journal of Tissue Culture Methods, 2001
Reproducible isolation and long term culture of epidermal keratinocytes from transgenic mouse lines is critically needed but most techniques have been unsuccessful. In this report we describe in detail a simplified method to isolate putative keratinocyte stem cells from newborn mouse skin and to maintain them for long term in culture. The cell cultures were established by enzymatically separating keratinocytes
In Vitro Cellular & Developmental Biology - Animal, 1992
We have developed a defined method for human epidermal keratinocyte culture. The minimally supplemented basal medium supported establishment of primary cultures from neonatal foreskin in a defined environment. It also supported serial cultivation and rapid expansion of cell number. Casein replaced serum for defined cryopreservation. Cells were serially cultivated in medium containing 0.08 mM calcium. The rate of cell division however remained high after addition of 1.8 mM calcium. The particulate transglutaminase activity of the cultures was low at confluence, even in the presence of 1.88 mM calcium, indicating an enrichment of the basal cell population. Culture with small amounts (0.3%) of chelated serum increased particulate transglutaminase activity approximately 2.2-fold in low calcium cultures and approximately 3.5-fold in high calcium cultures. A gradual reduction in growth rate of serum-treated cultures upon serial cultivation also indicated a depletion of cells with basal cell character. Bovine hypothalamic extract and cholera toxin were able to avert, in part, the differentiation-promoting effects of serum. Keratinocytes serially cultivated in the defined medium maintained the ability to develop normally into a morphologica/ly differentiated epidermis.