AIP Conference Proceedings 2193, 020012 (2019); https://doi.org/10.1063/1.5139332 2193, 020012
© 2019 Author(s).
Brief comparative study on the isolation and culture methods of human keratinocyte from skin tissue
Cite as: AIP Conference Proceedings 2193, 020012 (2019); https://doi.org/10.1063/1.5139332 Published Online: 10 December 2019
Retno Wahyu Nurhayati, Normalina Sandora, Suwarti, et al.
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Brief Comparative Study on the Isolation and Culture Methods of Human Keratinocyte from Skin Tissue
Retno Wahyu Nurhayati
1,2, Normalina Sandora
3,4,a), Suwarti
5,6, Raisa Nauli
4, Zakiyah Khoiriyah
6, Aditya Wardhana
71Stem Cells and Tissue Engineering Research Cluster, Indonesia Medical Education and Research Institute (IMERI), Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya No 6, Senen, Central Jakarta 10430
Indonesia
2Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya No 6, Senen, Central Jakarta 10430 Indonesia
3Faculty of Medicine, Universitas Riau, Pekanbaru, 28293, Indonesia
4Human Reproduction, Infertility, and Family Planning Research Center, IMERI, Faculty of Medicine, Universitas Indonesia, Jakarta 10430, Indonesia
5Indonesia Oxford Clinical Research Unit Laboratory (IOCRL), Eijkman Oxford Clinical Research Unit, Jl Salemba Raya No 6, Senen, Central Jakarta 10430 Indonesia
6Infectious Disease and Immunology Research Center, IMERI, Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya No 6, Senen, Central Jakarta 10430 Indonesia
7Plastic Surgery Division, Department of Surgery, Faculty of Medicine, Universitas Indonesia – Dr. Cipto Mangunkusumo General Hospital, Jakarta 10430, Indonesia
Corresponding author: a)[email protected]
Abstract. The healing of skin wounds involves various factors including keratinocytes, fibroblasts, extracellular matrix, nervous and vascular components of the surrounding tissue. During wound healing, keratinocytes migrate from basal area to cover the lesion area. The important role of keratinocytes in skin regeneration triggers interests to isolate and expand the cells in vitro. This study was attempted to compare briefly enzymatic and non-enzymatic keratinocytes isolation from adult skin. Non-enzymatic based keratinocyte isolation through explant culture allowed minimal sample preparation, however, the process required longer preparation time in which the target cells typically appears after 2 weeks culturing period. Meanwhile, enzymatic-based keratinocyte isolation required more complex procedure with relatively shorter preparation time. It is likely that explant method produced higher purity of keratinocyte stem cells as compared with direct digestion method, as analyzed from cytokeratin 14 expression.
Keywords: keratinocyte, skin, tissue, enzymatic digestion, cytokeratin
INTRODUCTION
Skin is an organ which protects the body from exogenous substances and barrier against the external environment.
Skin damage can occur as a result of cuts, burns, and resulting in permanent damage. If not repaired, any damaged of the skin may expose the body's tissues to mechanical damage and microbial infections [1]. Burn wound has the
potential to destruct all skin layers from epidermis up to hypodermis and other tissues such as blood vessel, neuron, tendon, and bone which can increase the chance of serious infection [2].
The healing of skin wounds involves various factors including keratinocytes, fibroblasts, extracellular matrix, nervous and vascular components of the surrounding tissue. Keratinocytes migrate from basal area to cover the lesion area, contact inhibition causes cell differentiation to stratified squamous keratinizing epidermal cells [3].
The skin is composed of two main layers: the epidermis and dermis, where the epidermis forms the outermost layer of the skin. Keratinocytes is the most abundant cell type in the epidermis [4]. Epithelial keratinocytes mainly compose the nonvascular layer, epidermis. These cells lead rapid and continuous proliferation and are thought to be continually regenerated from multipotent stem cells [5]. Keratinocytes move upward and leave the basal layer and, they differentiate into basal, spinous, granular, and cornified layers that correspond to specific stages [6]. Skin fibroblasts are located in the thicker dermis separated by the basement membrane below the epidermis. Skin fibroblast supply growth factors for keratinocytes, a property that important for early keratinocyte culture models. Thus, it has been possible to efficiently isolate both keratinocytes and fibroblasts from the same skin sample [4].
The keratinocytes also against pathogens in the skin in the first line, and are innate immune response [6].
Keratinocytes activate intracellular signaling pathways and afterwards, produce of a number of various inflammatory mediators including antimicrobial peptides, chemokines, and cytokines. Thus, because keratinocytes play a critical part in numerous biological processes, it is crucial to isolate keratinocyte.
This study was attempted to compare briefly enzymatic and non-enzymatic keratinocytes isolation from human skin from adult donors. Enzymatic and non-enzymatic (explant) methods were employed to isolate keratinocytes from epidermis.
MATERIAL AND METHODS Sample Preparation
The sample was the removed skin tissue from Sectio Cesarean patients 20-40 years old. The informed consent used the guidelines approved by the Ethics Committee from Faculty of Medicine Universitas Indonesia – Dr. Cipto Mangunkusumo General Hospital. Tissue was then preserved in the transfer medium containing Dulbecco's Modified Eagle Medium (DMEM; Life Technologies, USA), 10% Platelet Rich Plasma (PRP), 3x Antibiotic-antimycotic (Life Technologies), 1% Heparin (Pratama Nirmala, Indonesia).
Keratinocyte Isolation and Culture
Medium Preparation
Epilife complete medium: Epilife basal medium, 1x supplement S7 (Life Technologies) and 1x Antibiotic- Antimycotic. DMEM complete medium: DMEM, 10% fetal bovine serum, 1x AntibioticAntimycotic. Epilife-DMEM complete medium: equal ratio (vol/vol) of Epilife complete medium and DMEM complete medium.
Isolation and Culture Methods
Non-enzymatic/ Explant Method: Non-enzymatic isolation method was performed as described elsewhere [7].
Briefly, the skin tissue (2 × 2 cm2) was washed with DPBS Ca/Mg free on a petri dish and then submerged in povidone iodine for 10 minutes. The tissue was then washed with DPBS Ca/Mg free, clean up from fat and dermis, and then cut it into small pieces using surgical scalpel. The sample was trimmed and the epidermis was taken by a scalpel blade.
The sliced epidermis was then placed in collagen I coated dish. The explant was submerged with Epilife complete medium and then incubated at 37°C, 5% CO2. For uncoated dish, the explant was submerged with Epilife-DMEM complete medium [13].
Enzymatic Method. The fat-removed skin tissue (2 × 2 cm2) were soaked in Dispase solution (Life Technologies) and then incubated at 4°C 16 hours at 100 rpm. After incubation sample was washed with DPBS Ca/Mg free then the epidermis part was collected. The epidermis was treated with 5 mL Tryple Express (Life Technologies), then crushed using gentleMACS Dissociator (Miltenyi, Germany) following the manufacturer’s instruction. The obtained suspension sample was filtered using 100 μm cell strainer then centrifuged at 450 g for 10 minutes. Supernatant was
discarded and the pellet was dissolved with Epilife complete medium. The cell suspension was counted by a dye exclusion method with trypan blue.
Cell Counting
The trypan blue assay was performed to determine cell yield and viability upon isolation. Briefly, 10 µL of cell suspension was mixed with 10 µL of trypan blue solution (Life Technologies, USA). Then the cells were counted using a hemocytometer.
Keratinocyte Characterization
The cells at density 105 cells/tube were stained with FITC-conjugated anti-cytokeratin 14 (Abcam, USA) according to a manufacturer’s protocol. Keratinocyte cells were characterized using BD FACSAria III (BD Biosciences, USA) based on cytokeratin 14 expression.
RESULT AND DISCUSSION
Skin transplantation is remain challenging procedure because not only it should provide functional tissue of wound replacement but also meet the aesthetic criteria. Although mesenchymal stem cells are readily available and developed for skin transplantation, the formation of scar tissue is most likely to happen due to the tendency of MSCs to form fibroblasts rather than keratinocytes. Therefore, complementation with keratinocytes considered to play a pivotal role to improve the appearance of transplanted artificial skin. To support the complementation, decent keratinocytes procedure should be developed to have better cell yields with functional properties.
In this work, we performed two common keratinocyte extraction from adult skin tissue: explant culture and enzymatic digestion; and compared the feasibility for routine application. After fat was removed, the skin tissue was treated with a neutral protease (Dispase) [8-9] for detaching epidermis from dermis (Figure 1). This process is important to reduce fibroblast contamination during keratinocyte isolation.
(a) (b)
FIGURE 1. (a) Skin tissue before fat removal and dispase digestion. (b) Fat-free skin tissue separates into epidermis and dermis.
Bars indicate 1 cm.
Conventionally, keratinocytes are cultured with feeder dependent [10] or feeder free methods [11]. Feeder cells i.e. mouse 3T3 cells provide essential growth factor for improving cell proliferation. However, this method is unpractical and risky for clinical application. A study reported recombinant collagen coating and certain growth factors could substitute the need of feeder cells [12]. Xian et al [13] alternated collagen coating with DMEM with serum addition. Our results showed that combination of collagen coating and serum addition able to facilitate the keratinocyte growth. Keratinocytes were started to sprout from explant after 2 weeks of incubation (Figure 2a).
However, prolonged incubation in DMEM complete medium increased the growth of fibroblast cells (Figure 2b).
Therefore, medium replacement to Epilife complete medium immediately after keratinocyte sprouting would give better environment for keratinocytes to grow instead of fibroblast cells.
Epidermis Dermis
Dispase treatment
(a) (b)
FIGURE 2. (a) Morphology of keratinocytes in explant culture, (b) Morphology of fibroblast in explant culture.
In enzymatic method, Tryple Express was used to digest extracellular matrix. We noticed that without mechanical intervention, enzymatic method only released a low cell number of keratinocytes (< 2 × 104 viable cells). Therefore, a tissue dissociator was employed to physically crushed the digested tissue. As a result, the yield increased by 10-fold, in which 2.1 × 105 viable cells were collected.
Cytokeratins (CK) are important markers to determine the differentiation stage and location of human keratinocytes [14]. Mature keratinocytes, located in outermost skin layer, express CK5e. Committed keratinocytes, located in the middle epidermis layer, express CK1 and CK10. Meanwhile, keratinocyte stem cells, located in basal layer, express CK14, CK15 and CK5. Keratinocyte stem cells are actively proliferating and become valuable resources for skin transplantation. In this study, we evaluated the CK14 expression of keratinocytes collected by enzymatic digestion and explant methods. Our results in figure 2 showed after 20-day culture, explant method produced higher CK14+ cell population (37.7% CK14+ cells) as compared with cells obtained from enzymatic digestion method (19.3%
CK14+). This result suggested that culture condition specifically promoted keratinocyte stem cells and repressed other cell types.
In conclusion, non-enzymatic based keratinocyte isolation through explant culture allowed minimal sample preparation, however, the process required longer preparation time in which the target cells typically appeared after 2 weeks culturing period. Meanwhile, enzymatic-based keratinocyte isolation required more complex procedure with relatively shorter preparation time (~ an hour). It is likely that explant method produced higher purity of keratinocytes as compared with direct digestion method, as analyzed from cytokeratin 14 expression.
FIGURE 3. The population of keratinocyte stem cells as analyzed from CK14 expression.
CONCLUSION
This study was attempted to compare briefly enzymatic and non-enzymatic keratinocytes isolation from adult skin.
Non-enzymatic based keratinocyte isolation through explant culture allowed minimal sample preparation, however, the process required longer preparation time in which the target cells typically appears after 2 weeks culturing period.
Meanwhile, enzymatic-based keratinocyte isolation required more complex procedure with relatively shorter preparation time. It is likely that explant method produced higher purity of keratinocyte stem cells as compared with direct digestion method, as analyzed from cytokeratin 14 expression. Alternatively, it is interesting to see whether enzymatic digestion method culture within the same period of time lead to similar yields of explant method. Hence, the keratinocytes CK14+ cells profile is not per se due to isolation method but also resulted of cells proliferation within a such period of time. Further exploration on this hypothesis require more experimental procedures.
ACKNOWLEDGMENTS
This research is funded by Penelitian Terapan Unggulan Perguruan Tinggi (PTUPT) Universitas Riau grant from Indonesian Ministry of Research and Technology and Higher Education lead by Dr Normalina Sandora (Contract No:
792/UN.19.5.1.3/PT.01.03/2019).
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