A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Summary

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Abstract

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Introduction

The goal was to develop a simple and efficient protocol to isolate primary human keratinocytes (HKCs) from adult tissues, especially for clinical applications. Skin epidermal stem cells, localized in the basal layer of the skin, possess a high potential to proliferate and differentiate and provide keratinocytes to maintain the functions of the skin^1,2,3,4. HKCs isolated from skin tissues are widely used for skin tissue engineering and regeneration purposes, especially in the repair of damaged skin and in gene therapy for clinical applications^5,6. The key issue for HKC-based applications is to efficiently isolate and expand large numbers of HKCs with high potential in vitro^7,8. Although various research groups have developed methods to produce cultures of stem-like HKCs, these methods are sometimes time-consuming and complicated to perform and have other limitations, such as low cell yields and being limited by the type of skin specimen used⁹. For instance, the traditional method to isolate HKCs from skin tissues involves a two-step enzymatic digestion with a separation of the epidermis from the dermis⁶. That method usually works well for neonatal tissues, but it becomes very difficult when used to isolate cells from adult tissues.

Y-27632, an inhibitor of Rho-associated protein kinase (ROCK), has been reported to significantly enhance the efficiency of epidermal stem cell isolation and colony growth^10,11,12. In a previous study, we discovered that Y-27632 facilitates the clonal growth of epidermal cells but reduces the yield of dermal cells by differentially controlling the expression of adhesion molecules¹³. We also established a new conditioned inoculation medium, called G-medium, which supports the growth and yield of primary epidermal cells. By combining G-medium with Y-27632, this novel method can spontaneously separate epidermal and dermal cells after enzyme digestion, thus removing the step of epidermis-dermis separation^13,14. Based on previous reports, we now describe the detailed procedure of this new method to isolate HKCs from adult skin tissue.

Protocol

Human tissues used in this protocol have been handled according to the guidelines of the Institution's human research ethics committee (NO.2015120401, date: May 12, 2015).

1. Preparations

1. Collect fresh adult abdominal skin tissues discarded from plastic surgery at the hospital in a 50-mL tube with 10 mL of ice-cold Dulbecco's modified Eagle medium (DMEM). The specimen can be kept at 4 °C for up to 72 h without significantly affecting the cell viability.
2. Prepare the reagents and culture medium as described below.
   1. Add 1 mL of penicillin (100 U/mL) and streptomycin (100 mg/L) to 50 mL of phosphate-buffered solution (PBS) to prepare the washing solution.
   2. Prepare two sets of enzyme digestion solutions: (1) prepare 50 mL of dispase (2.5 mg/mL in DMEM) and 50 mL of type I collagenase (2.5 mg/mL in DMEM) separately for the conventional method; and (2) prepare 50 mL of a mixture of dispase and type I collagenase (dissolve 125 mg of dispase powder and 125 mg of type I collagenase powder in 50 mL of DMEM) for the new method.
      NOTE: All enzyme solutions should be filtered with a 0.22-µm strainer and kept at4 °C.
   3. Prepare digestion solutions for both methods: 0.05% trypsin and 0.25% trypsin were commercially purchased. Prepare a 10 mg/mL DNase I solution by dissolving 50 mg of DNase I powder in 5 mL of PBS, filter it with a 0.22-µm strainer, and store it at -20 °C.
   4. Prepare 500 mL of medium to neutralize the enzymatic digestion: DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.
   5. Prepare 500 mL of inoculation medium (growth-factor-containing medium, called G-medium): DMEM/F12 (3:1) medium containing 1% penicillin/streptomycin, 40 µg/mL fungizone, 40 ng/mL fibroblast growth factor 2 (FGF2), 20 ng/mL epidermal growth factor (EGF), and 2% B27 supplement.
   6. Pretreat 100-mm cell culture dishes with 2 mL of coating matrix containing type I collagen for 30 min at room temperature.

2. Conventional Method

1. Skin tissue pretreatment
   1. Take 1.5 cm² of skin tissue and wash it 1x with 10 mL of PBS; then, rinse it with 10 mL of 70% ethanol for 30 s and incubate it 2x with 10 mL of washing solution (PBS containing 2% penicillin and streptomycin), 5 min each.
      NOTE: All washes are performed in 100-mm cell culture dishes inside a laminar flow hood.
   2. Trim the tissue with sterile scissors to remove the subcutaneous fat layer in a 100-mm cell culture dish and, then, weigh the skin tissue (the weight is 1 g in this protocol).
      NOTE: Sufficient trimming is very crucial for the efficient separation of the epidermis from the dermis. The yellowish fat layer can be easily distinguished visually.
   3. Transfer the tissue to another 100-mm sterile dish with the dermis side down and, then, cut the skin tissue into 3- to 4-mm-wide strips using a scalpel blade.
      NOTE: The dermis side with the fat layer is easily recognized visually.
   4. Add 10 mL of 2.5 mg/mL dispase solution to the skin tissues in a 100-mm culture dish and, then, incubate the dish overnight at 4 °C (no more than 20 h).
      NOTE: The amount of dispase solution can be calculated by using 10 mL of dispase solution for the digestion of each gram of tissue.
2. Separation of the epidermis from the skin tissue
   1. On the second day, peel off the epidermis from the dermis using fine tweezers.
      NOTE: If it is difficult to peel off the epidermis, the dispase digestion was not sufficient, which is usually due to insufficient trimming of the tissue. In this case, the tissue needs a longer incubation time.
   2. Mince the peeled epidermis with 500 µL of cell culture medium into a tissue slurry using scalpel blades; then, resuspend it with 10 mL of 0.25% trypsin in a 50-mL tube and incubate it 20 min in a water bath at 37 °C, with shaking.
3. Collection and culturing of primary cells
   1. Neutralize the trypsin activity by adding an equal volume of neutralization solution (10 mL in this protocol).
   2. Dissociate the epidermal cells by pipetting the solution up and down 20x with a 10-mL serological pipette and pass the cell solution through a 100-µm cell strainer to remove any residual tissue debris.
   3. Centrifuge the cell solution at 200 x g for 5 min after filtration; resuspend the cell pellet in neutralization medium for washing and, then, centrifuge it again at 200 x g to obtain the cell pellet.
   4. Resuspend the cell pellet in 10 mL of low-calcium, serum-free keratinocyte medium (SFM). Take 10 µL of this solution to count the total cell number and, then, plate about 2 x 10⁶ cells with 10 mL of SFM into 100-mm cell culture dishes pretreated with coating matrix (containing type I collagen).
      NOTE: Coating is a necessary step for the conventional method.
   5. Change the culture medium every 2 d.
      NOTE: Check the cell adhesion under a microscope before and after changing the culture medium.
   6. Passage the cells when they reach about 80% confluence.
      NOTE: All culture dishes are pretreated with the coating matrix.

3. The New Method

1. Skin tissue pretreatment
   1. Collect the tissue as described above (step 2.1) for the conventional method.
   2. Wash the skin tissue 1x with 10 mL of PBS and, then, weigh it in a sterile plastic container by an electronic scale (the weight is around 1 g in this protocol).
      NOTE: The minimum weight of tissue needed for this protocol is 0.1 g, since it is difficult to obtain a sufficient number of cells if the tissue specimen used is too small. There is no maximum weight of tissue for this protocol, which ultimately depends on the handling capacity of the operator.
   3. Use forceps to rinse the skin tissue with 10 mL of 70% ethanol for 30 s.
   4. Incubate the tissue 2x with 10 mL of washing solution (prepared in step 2.1.1), for 5 min each time.
      NOTE: All the washing steps noted above are performed in 100-mm cell culture dishes inside a laminar flow hood (a tissue culture hood).
   5. Transfer the tissue to another 100-mm cell culture sterile dish.
   6. Using scalpel blades, mince the tissue thoroughly into a tissue slurry.
   7. Add 200 µL of PBS every 5 min to keep the tissues wet.
      NOTE: Take around 15 min for steps 3.1.5 - 3.1.7. All the above steps are performed at room temperature.
2. Digestion of skin tissue
   1. Transfer the homogenized tissue solution into a 50-mL tube. Add 10 mL of enzyme mixture for every 1 g of skin tissue. Mix the enzymes thoroughly with the homogenized tissues.
   2. Incubate the mixture with shaking in a water bath at 37 °C for 1 h.
   3. Add a 1/5 volume of 0.25% trypsin (2.5 mL in this protocol) to the digestion mixture for another 30 min in a water bath at 37 °C.
   4. Add DNase I solution to the enzyme mixture at a 1:100 (v/v) ratio (250 µL in this protocol) and incubate it for 5 min at room temperature.
3. Collection and culturing of primary cells
   1. Stop the digestion process by adding 12.5 mL of neutralization medium in a 1:1 ratio. Pipette the solution up and down for about 20x, using a 10-mL serological pipette to dissociate the cells.
   2. Filter the dissociated cells through a 100-µm cell strainer to remove the tissue debris. Centrifuge the supernatant at 200 x g for 5 min. Observe the pellet at the bottom of the tube.
   3. Discard the supernatant and wash the cell pellet with 10 mL of neutralization medium, and centrifuge again at 200 x g for 5 min to collect the cells.
   4. Resuspend the cell pellet in 10 mL of inoculation medium (G-medium from step 1.2.5) with 10 µM of Y-27632.
   5. Take 10 µL of the solution to count the total cell number and, then, plate about 2 x 10⁶ cells with 10 mL of G-medium into a 100-mm cell culture dish.
      NOTE: The precoating step is not necessary for the new method, and no removal of the dermal layer is required either.
   6. After 3 d, replace the inoculation medium with low-calcium SFM medium. Change the culture medium every 2 d.
      NOTE: Check the cell adhesion before and after changing the medium.
   7. Passage the cells when they reach about 80% confluence.
      NOTE: If necessary, pretreat the cells with 0.05% trypsin for 2 min and wash the cells with PBS to remove contaminating dermal cells before trypsinizing the epidermal cells.

4. Cell Passaging

1. Remove the medium, wash the cells with PBS, and, then, add 2 mL of 0.05% trypsin (per each 100-mm dish).
2. Incubate the cells in an incubator (37 °C with 5% CO₂) for 5 min.
3. Check the cells using a microscope to make sure that all cells have detached from the culture dish.
4. Add 8 mL of DMEM with 10% FBS to neutralize the enzymatic reaction and, then, collect the cells in a 15-mL tube.
5. Centrifuge at 200 x g for 5 min to obtain the cell pellet.
6. Remove the supernatant slowly and, then, resuspend the cell pellet with 10 mL of SFM medium and count the number of cells.
7. Plate 1 x 10⁶ cells with 10 mL of SFM into each 100-mm cell dish.
8. Change the medium every 2 d.

Results

Schematic diagrams of the new method (Figure 1A) and the conventional method (Figure 1B) are presented in Figure 1. The conventional method is a two-step digestion, which requires a 2-day procedure. By contrast, the new method is a one-step digestion, which takes around 3 hours to perform. Importantly, the one-step new method can obtain two populations (epidermal and dermal cells) at the same time, w...

Discussion

Cultured primary HKCs have been widely utilized to treat wounds in clinics for more than three decades and, since that time, it has been always important to efficiently obtain sufficient numbers of cells for clinical applications in a timely manner. Therefore, in practice, the conventional isolation method, which requires the separation of the epidermis from the dermis, makes it difficult to meet these demands, due to the low yield of cells and the low ability to passage adult cells. Here, we describe a new simple method...

Materials

Name	Company	Catalog Number	Comments
Countess automated cell counter	Invitrogen Inc.	C10227	Automatic cell counting
CO2 Incubator	Thermo Scientific	51026333	For cell incubating
Sorvall ST 16R Centrifuge	Thermo Scientific	75004380	Cell centrifuge
Constant Temperature Shaker	Shanghai Boxun	150036	For water bath
Electronic Scale	Harbin Zhonghui	1171193	For tissue weighing
Cell Culture Dish	Eppendorf	30702115	For cell culture
50ml Centrifuge Tube	KIRGEN	171003	For cell centrifuge
Cell Strainer	Corning incorporated	431792	Cell filtration
Phosphate buffered solution	Solarbio Life Science	P1020-500	Washing solution
DMEM	Thermo Scientific	C11995500	Component of neutralization medium
Defined K-SFM	Life Technologies	10785-012	Epidermal cells culture medium
Penicillin Streptomycin	Thermo Scientific	15140-122	Antibiotics
Fetal Bovine Serum	Biological Industries	04-001-1AC5	Component of neutralization medium
0.05% Trypsin	Life Technologies	25300-062	For HKC dissociation
0.25% Trypsin	Beijing Solarbio Science & Technology	T1350-100	For HKC dissociation
Coating Matrix Kit	Life Technologies	R-011-K	For coating matrix
Dispase	Gibco	17105-041	For HKC isolation
Collagenase Type I	Life Technologies	17100-017	For HKC isolation
Deoxyribonuclease I	Sigma	9003-98-9	For HKC isolation
F12 Nutrient Mix, Hams	Life Technologies	31765035	Component of G-medium
B27 Supplement	Life Technologies	17504044	Growth factor in G-medium
FGF-2 Millipore	Merck Biosciences	341595	Growth factor in G-medium
Y-27632	Sigma-Aldrich	Y0503	ROCK inhibitor
Fungizone	Gibco	15290026	Preparation for G-medium
EGF Recombinant Human Protein	Gibco	PHG0311	Growth factor in G-medium
Cell Counting Kit-8	Thermo Scientific	NC9864731	cell proliferation and cytotoxicity assays
Mouse Anti-Human Cytokeratin5	Hewlett-Packard Development Company	MA-20142	For immunofluorescence staining to check differentiation marker of HKCs
Rabbit Anti-Human Loricrin	Covance	PRB-145p	For immunofluorescence staining to check differentiation marker of HKCs
Mouse anti-human Vimentin	Cell Signaling Technology	3390	For immunofluorescence staining of dermal fibroblasts
Integrin α6(GOH3)	Santa Cruz	SC-19622	flow cytometry analysis of HKCs
Rat IgG2a FITC	Santa Cruz	SC-2831	negative control antibody of α6-integrin  in flow cytometry analysis