Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

Summary

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

Abstract

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.

Introduction

The human gingiva, the first line defense structure that protects periodontal tissue, is not only a physical and chemical barrier¹, but also secretes different classes of inflammatory mediators that participate in immune responses and constitute an immune barrier^2,3. The gingival epithelium plays an important role in the repair and regeneration of periodontal tissue. Therefore, studying the defense and immunity of the gingival epithelium is of great significance for understanding the occurrence, diagnosis, and treatment of periodontitis. The isolation and culture of gingival epithelial cells from human gingival tissue is the first step required for studying the gingival epithelium. Such a procedure requires basic operations such as producing seed cells for tissue engineering, in vitro models of periodontal associated diseases, and materials for repairing periodontal defects.

Primary gingival epithelial cells are characterized by a low division rate in vitro⁴, researchers have been looking for an optimal isolation and cultivation method for decades. To date, two different techniques, a direct explant method and an enzymatic method, are commonly used in laboratories to obtain primary gingival epithelium cells in vitro^4,5. The direct explant method has advantages such as the requirement of a lower amount of tissue specimens and simple isolation procedure, but it has the disadvantages of longer culture time and susceptibility to contamination⁵. Although the enzymatic method shortens the culture time required, the efficiency is relatively low and varies depending on the enzymes and medium used. Kedjarune et al.⁶ showed that the direct explant method, which requires more time before subculture (2 weeks), appeared to be more successful for culturing gingival epithelial cells compared to the enzymatic method. However, comparing these two methods, Klingbeil et al.⁷ found that the enzymatic method had the best results for primary cultures of oral epithelial cells, and it was possible to obtain the optimal cell yield within the shortest time period (11.9 days versus 14.2 days).

Therefore, it was important to develop a more convenient and effective method for the isolation and culture of oral epithelial cells⁴. We previously reported that adding Y-27632, an inhibitor of Rho-associated protein kinase (ROCK), simplifies the isolation procedure of human primary epidermal cells and keratinocytes from adult skin tissues^8,9,10. We developed G-medium, a new conditioned inoculation medium that spontaneously separates epidermal from dermal cells and supports the growth and yield of primary epidermal cells^8,9,10. In the present study, we developed a new serum-free isolation and culture technique for gingival epithelial cells by combining G-medium with Y-27632. In essence, our method is based on a simplification of the traditional two-step enzymatic method, so we compared our new method with the direct explant method. This modified enzymatic method significantly shortens the time required to separate gingival epithelial cells from gingival tissue and increases the efficiency of culturing gingival epithelial cells.

Protocol

Human tissues used in this protocol are fresh adult gingival tissues discarded from extractions of impacted teeth in the Maxillofacial Surgery Department according to the guidelines of the Institution's Human Research Ethics Committee (Protocol No. GR201711, Date: 02-27-2017).

1. Preparations

1. Collect fresh adult gingival tissues in 15 mL tubes containing 10 mL of phosphate-buffered saline (PBS) supplemented with 3% penicillin/streptomycin (P/S) and keep the tissues at 4 °C.
   NOTE: Treat the tissues as detailed below within 24 h after excision.

2. Prepare reagents and culture medium

1. Prepare the washing solution: 3% P/S. Mix 50 mL of PBS with 1.5 mL of penicillin (100 U/mL) and 1.5 mL of streptomycin (100 mg/L).
2. Prepare 500 mL of growth-factor-containing medium (called G-medium): DMEM/F12 (3:1) medium containing 1% P/S, 2% B27 supplement, 20 ng/mL of epidermal growth factor (EGF), 40 ng/mL of fibroblast growth factor 2 (FGF2), and 40 µg/mL of fungizone.
3. Prepare enzyme digestion solutions for the new method: Dissolve 125 mg of Dispase powder and 125 mg of Type I collagenase powder in 50 mL of DMEM.
   NOTE: Filter all the enzyme solutions through a 0.22 µm strainer and store at 4 °C.
4. Prepare 500 mL of the medium to neutralize the enzymatic digestion: 10% fetal bovine serum (FBS) and 1% P/S in the DMEM medium.

3. Direct explant method

1. Gingival tissue pretreatment
   1. Wash gingival tissue with 5 mL of 75% ethanol for 30 s. Then, rinse twice with 5 mL of the washing solution (step 2.1) for 5 min.
      NOTE: Perform all washes in cell culture dishes with a diameter of 50 mm.
2. Gingival epithelial cell culture
   1. Cut the gingival tissue into 1 mm³ pieces and scatter them in a 100 mm cell culture dish. Use fine pointed forceps and ophthalmic scissors for the cutting operations.
   2. Add the G-medium (step 2.2) to the culture dish. Then, incubate the culture dish in a 5% CO₂ incubator at 37 °C. Use an inverted microscope (40x) to examine the tissues every 24 h.
   3. Remove the gingival tissue pieces when the epithelial cells have propagated to a 2-5 mm in diameter around the tissue pieces. Change the G-medium every 2-4 days.

4. The new modified enzymatic method

1. Gingival tissue pretreatment
   1. For this, follow the same steps as described for the direct explant method, step 3.1.1.
2. Digestion of gingival tissue
   1. Cut the gingival tissue into tiny pieces, approximately <1 mm in size, with two sterilized blades. Repeat the process of shredding with two surgical blades.
   2. Add 1 mL of 2.5 mg/mL dispase + collagenase solution to a 1.5 mL centrifuge tube containing the gingival tissue pieces, and then incubate the tube for 15-20 min in an incubator at 37 °C.
   3. When the tissue becomes transparent and flocculent, add 1 mL of the neutralization solution to terminate the digestion. Thoroughly mix the solution by pipetting up and down 10-15 times. Pass the solution through a 100 µm mesh filter.
   4. Centrifuge at 200 x g for 5 min.
   5. Remove the supernatant and resuspend the cell pellet at the bottom in 10 mL of growth-factor-containing medium (step 2.2). Transfer the cell suspension to a 100 mm cell culture dish. Add 10 µM of Y-27632 to the cell culture dish.
   6. Culture the cells at 37 °C in a 5% CO₂ incubator. Replace the old G-medium with the fresh G-medium every 2 days. Observe the cells on Day 1 and Day 3.

5. Cell passaging

1. Take the dish out of the incubator, remove the spent medium and wash twice with PBS. Add 2 mL of 0.05% trypsin for each 100 mm dish.
   NOTE: Shake the dish to make sure there is enough contact between the Trypsin solution and the dish bottom.
2. Leave the dish in an incubator around 5 min at 37 °C for the digestion process.
3. Use a microscope (40x) to examine the cells and make sure that most cells have separated from the bottom of the dish.
4. Add 2 mL of the neutralization solution to stop the digestion and transfer the cells into a 15 mL tube. Pipette up and down 10-15 times. Centrifuge the cells at 200 x g for 5 min.
5. Remove the supernatant slowly. Resuspend the cells with 10 mL of G-medium and count the number of cells.
6. Add about 1 x 10⁶ cells in 10 mL of G-medium and 10 µM Y-27632 to each 100 mm dish.
7. Renew the G-medium and Y-27632 every 2 days. View the cells on Day 1 and Day 5.

Results

Figure 1 shows a schematic diagram of the direct explant method and the modified enzymatic method. The direct explant method does not need any digestive enzyme during the whole process. In contrast, the traditional enzymatic method usually needs two sets of digestive enzymes, dispase and collagenase, to separate the epithelial sheet from the underlying fibroblast layer, and then trypsin to release the epithelial cells into suspension. Our new method omits the step of separation and is a simp...

Discussion

The gingival tissue is a key structure that maintains periodontal integrity and health. Gingival epithelial cells have significant roles in the repair and regeneration of periodontal tissue and can be used in scientific research and clinical applications and related fields, including oral biology, pharmacology, toxicology, and oral mucosa deficiencies¹⁸. Therefore, it is necessary to develop a stable and efficient method to harvest oral epithelial cells¹⁹. Primary epithelia...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Names			Abbreviations & Comments
Countess automated cell counter	Shanghai Ruiyu Bio-science&Technology Co.Ltd.	BBA0218AC	Automatic cell counting
CO2 Incubator	Thermo Scientific	51026333	For cell incubation
Sorvall ST 16R Centrifuge	Thermo Scientific	75004380	Cell centrifuge
Cell Culture Dish	Eppendorf	30702115	For cell culture
50 ml Centrifuge Tube	KIRGEN	171003	For cell centrifugation
1.5 ml microcentrifuge Tubes	KIRGEN	190691J	For cell digestion
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	Gingival epithelial  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 HGGEPCs dissociation
Dilution Medium	Life Technologies	50-9701	For coating matrix
Dispase	Gibco	17105-041	For HGGEPCs isolation
Collagenase Type I	Life Technologies	17100-017	For HGGEPCs 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	Gene Operation	IAD1011	ROCK inhibitor
Fungizone	Gibco	15290026	Preparation for G-medium
EGF Recombinant Human Protein	Gibco	PHG0311	Growth factor in G-medium
Cell Counting Kit-8	Dojindo Molecular Technologies	CK04	For Cell proliferation assay
Rabbit Anti-Human CK18	Abcam	ab82254	For immunofluorescence staining to check differentiation marker of HGGEPCs
Rabbit Anti-Human Cytokeratin10	Abcam	ab76318	For immunofluorescence staining to check differentiation marker of HGGEPCs
Mouse anti-human Vimentin	Cell Signaling Technology	3390	For immunofluorescence staining of Gingival fibroblasts
Rabbit Anti-Human pan-ck	BD	550951	For immunofluorescence staining to check differentiation marker of HGGEPCs
rabbit anti-Ki67	Abcam	15580	For immunofluorescence staining to check differentiation marker of HGGEPCs
rabbit anti-p63	Biolegend	619002	For immunofluorescence staining to check differentiation marker of HGGEPCs
rabbit anti-p75NGFR	Abcam	ab52987	For immunofluorescence staining to check differentiation marker of HGGEPCs