Isolation of Epithelial Cells from Human Dental Follicle

Podsumowanie

The dental follicle contains an epithelial population and mesenchymal cells. The epithelial population was selected from the heterogeneous dental follicle cell population by providing a distinct culture medium. Epithelial cells survived and formed colonies in a serum-free medium.

Streszczenie

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was performed on the day of DF harvest. The DF was washed three times with DPBS and then dissected with tissue scissors until the tissue had a pulpy or squishy consistency. Single-cell populations were pelleted by centrifugation and washed with keratinocyte serum-free medium. Heterogeneous cell populations were distributed in a culture dish. Keratinocyte serum-free medium was used to select the epithelial cells. The culture medium was changed daily until no floating debris or dead cells were observed. Epithelial cells appeared within 7-10 days after cell population distribution. Epithelial cells survived in serum-free medium, while α-modification minimal essential medium supplemented with 10% fetal bovine serum allowed the proliferation of mesenchymal-type cells. The DF is a tissue source for the isolation of dental epithelial cells.

The purpose of this study was to establish a method for the isolation of epithelial cells from human DF. Periodontal ligament (PDL) was used for the isolation of human dental epithelial cells. Procuring epithelial cells from human PDL is not always successful due to the small tissue volume, leading to low numbers of epithelial cells. DF has a larger volume than PDL and contains more cells. DF can be a tissue source for the primary culture of human dental epithelial cells. This protocol is easier and more efficient than the isolation method using PDL. Procuring human dental epithelial cells may facilitate further studies of dental epithelial-mesenchymal interactions.

Wprowadzenie

Tooth formation begins with the invagination of the oral epithelium¹. According to the tooth developmental stage, the oral epithelium has different names, including inner and outer enamel epithelium, cervical loop, and Hertwig's epithelial root sheath (HERS). The epithelial compartments communicate with the surrounding mesenchymal cells. Epithelial-mesenchymal interactions regulate tooth formation and tissue regeneration. Procuring dental epithelial cells, such as oral keratinocytes and Hertwig's epithelial root sheath cells (HERSCs), is crucial for the study of dental epithelial-mesenchymal interactions².

Rodent-derived dental epithelial cells are isolated from the epithelial structure, such as the HERS. Li and colleagues isolated and immortalized rat molar-derived HERSCs after harvesting the apical portion of the developing tooth germs from 8-day-old rats³. The HERS was separated from apical tissue under magnification. Considering the tooth developmental stage and age, harvesting the HERS from humans is nearly impossible because of ethical issues: a developing tooth germ needs to be removed from a young child to harvest the human HERS. Immature tooth germs are rarely extracted. Human dental epithelial cells can be isolated from the gingiva and periodontal ligament (PDL). Epithelial structure-derived cells participate in tooth formation together with mesenchymal components and might be more suitable for the study of dental epithelial-mesenchymal interactions than oral keratinocytes. Epithelial cell rests of Malassez (ERM) are HERS-derived epithelial remnants and reside in small numbers in the PDL⁴. Studies report the isolation of human HERSCs from PDL⁵. However, harvesting human HERSCs from PDL tissue is not always successful because of the scarcity of the epithelial population in this location^5,6.

Although rodent-derived HERSCs are maintained in serum-containing media^3,7, human DF-derived epithelial cells are cultured with serum-free media similar to other human epithelial cells, such as normal human epidermal keratinocytes and normal human oral keratinocytes^8,9. This implies physiologic or functional differences between rodent dental epithelial cells and human dental epithelial cells. Understanding the mechanism regarding dental epithelial-mesenchymal interactions might contribute to the development of clinical applications, including periodontal reattachment during replantation, periodontal regeneration in periodontal disease, pulp-dentin complex regeneration, and bio-tooth generation. Considering the characteristics of translational research, human dental epithelial cells may be more appropriate than rodent dental epithelial cells for the study of epithelial-mesenchymal interactions.

The human DF is a loose connective tissue and often resides in an impacted tooth. The DF contains mesenchymal precursors¹⁰. However, to our knowledge, no study has reported the isolation of epithelial cells from dental follicles before 2021. Oh and Yi reported the isolation of epithelial cells from human DF in 2021⁸. The epithelial phenotype was confirmed by western blotting and morphologic analysis. Analysis of the origin of DF-derived epithelial cells demonstrated similar results with other studies. DF-derived epithelial cells were neither endothelial nor hematopoietic^5,11, and Oh and Yi suggested naming these cells as DF-HERSCs. The DF has a larger volume than the PDL, and more epithelial cells can be isolated from the DF. This enhances the emergence of epithelial colonies and results in a high success rate in harvesting epithelial cells from DF. This study suggests using the DF as a tissue source for the isolation of dental epithelial cells.

In the present study, single cells were isolated from the DF according to previously described procedures^10,12. The DF contains heterogeneous cell populations, and several cell types could be present at the early stage of the procedure. Morsczek and colleagues isolated DF-derived mesenchymal stem cells¹⁰. We hypothesized that the DF contains epithelial cells and that only epithelial cells can survive under serum-free conditions. This study differs from that of Morsczek et al. in terms of the selection of the epithelial population and the inhibition of mesenchymal cells. The selection was performed using keratinocyte serum-free media (SFM), which allows epithelial cell proliferation and inhibits mesenchymal cell proliferation. This study originated from a report by Oh and Yi⁸. The aim of this study was to describe details of the method used in that report for the isolation of epithelial cells from human DF.

Protokół

This study was approved by the Institutional Review Board of Kyung Hee University Hospital at Gangdong (IRB approval no. KHNMC 2017-06-009).

1. Collect DF

NOTE: Patients gave informed consent before surgery for the removal of a mature or immature impacted third molar. Patients with the following disease were excluded: diabetes, hypertension, tuberculosis, hepatitis, acquired immunodeficiency syndrome. Pregnant women were also excluded.

1. Harvest DF during the surgical extraction of impacted third molars (Figure 1A).
   NOTE: Surgery was performed by an oral maxillofacial surgeon. Cooperation is required with a dentist for tissue collection.
2. Store DF in Dulbecco's phosphate-buffered saline (DPBS) with 3% penicillin-streptomycin at 4 °C before use. Perform isolation procedures on the day of DF harvest (Figure 1B).

2. Isolate single-cell populations from DF

1. Prepare stock solutions of collagenase type I and protease in DPBS so that final concentrations of collagenase type I and protease are 1 mg/mL and 2.4 mg/mL, respectively.
2. Prepare three 50 mL washing tubes with 20 mL of DPBS per tube. Label the tubes as 1, 2, and 3.
3. Wash the DF in the washing tubes sequentially. Hold the DF with tweezers and place the DF in tube 1. Take the DF out of tube 1 and place it in tube 2. Take the DF out of tube 2 and place it in tube 3. Gently shake DF 10-15 times in each tube. After washing three times, place the DF in a 60 mm culture dish (Figure 2A).
4. Mince the DF with tissue scissors (Figure 2B). Transfer only a small portion to the culture dish to minimize tissue loss. Repeat the cutting until the DF has a pulpy or squishy appearance (Figure 2C).
5. Mix 1 mL of collagenase type I and 1 mL of protease solution in a 15 mL conical tube to obtain final concentrations of 1 mg/mL and 2.4 mg/mL, respectively.
6. Transfer the minced DF into the 15 mL conical tube from step 2.5. Gently shake and incubate the tube for 1 h at 37 °C.
7. Add 5 mL of 0.05% trypsin-EDTA to the tube, shake, and incubate the tube for 15 min at 37 °C.
8. Prepare keratinocyte medium containing keratinocyte SFM 10% fetal bovine serum (FBS). Add 3 mL of keratinocyte medium into a new 50 mL conical tube. Place a 40 µm strainer on top of this tube and use a 10 mL serological pipette to aspirate the supernatant from step 2.6 and pass it through the strainer.
   NOTE: Minimize the incorporation of digested tissue remnants while taking the supernatant. Remove the tissue remnants with a 40 µm strainer to minimize failure. Tissue remnants may pass through 70 µm strainers and hinder colony formation. FBS in the keratinocyte medium will inactivate the enzymes.
9. Repeat steps 2.7 and 2.8.
10. Transfer the collected suspension to a 15 mL conical tube.
11. Centrifuge the 15 mL conical tube at 288 × g for 3 min at 4 °C. Remove the supernatant.
    NOTE: Be careful to avoid any accidental removal of pelleted cells, which are mostly invisible.
12. Add 3 mL of keratinocyte SFM and wash the cells twice with a 1 mL pipette with centrifugation as in step 2.11.
13. Plate the single-cell population into a 60 mm culture dish using keratinocyte SFM. Maintain the culture dish with a final volume of 5 mL in a humidified atmosphere of 5% CO₂ at 37 °C. Change the culture medium daily until no tissue or cell debris are observed.
    NOTE: Remove floating debris in the culture medium by changing the medium. Delayed removal of tissue debris or dead cells has an unfavorable effect on the primary culture.
14. Examine the plate from step 2.13 to check the growth of epithelial cells (Figure 3A).
    NOTE: Epithelial cells grow within 7-10 days after plating single-cell populations.
15. Expand the epithelial colonies and subculture the cells.
    1. Aspirate the medium and wash the bottom of the culture plate once with 3 mL of keratinocyte SFM.
    2. Add 2 mL of cell dissociation protease and incubate in a humidified atmosphere of 5% CO₂ at 37 °C for 3 min.
       NOTE: Repeat step 2.15.2 if cell detachment is not effective.
    3. Add 2 mL of keratinocyte medium to the culture plate and transfer the contents in a 15 mL conical tube.
       NOTE: Adding more keratinocyte medium is not required if step 2.15.2 is repeated.
    4. Centrifuge the 15 mL conical tube at 288 × g for 3 min at 4 °C.
    5. Remove the supernatant and wash the cell pellet two times with 3 mL of keratinocyte SFM.
    6. Plate the cells in a 100 mm culture dish using keratinocyte SFM (final volume of 10 mL per 100 mm dish).
16. Prepare and store cell stocks in a liquid nitrogen tank.
    1. Harvest cells as described in steps 2.15.1-2.15.5.
    2. Distribute the cells into cryovials in 1 mL of freezing medium (80% keratinocyte SFM, 10% dimethylsulfoxide, 10% FBS).
    3. Place the vials in a freezing container and store them at -80 °C for 24 h.
    4. Transfer the vials from the deep freezer to a liquid nitrogen tank.

Wyniki

DF harvesting
Surgery was performed by an oral maxillofacial surgeon. Human-derived materials, including the tooth fragment, gingival tissue, and DF, were collected by a surgeon (Figure 1A). The DF might be attached to the tooth fragment. An oral maxillofacial surgeon will be able to identify the DF. Cooperation and communication with the surgeon are required for tissue collection. The DF is an irregularly shaped membrane-like tissue. Gingival tissue has a keratinized ...

Dyskusje

This protocol includes critical steps. Harvesting single-cell populations is essential for the successful isolation of epithelial cells from DF. We sought to isolate epithelial cells from the DF based on our hypothesis that there are more epithelial cells in the DF. The mincing procedure enhances the detachment and release of cells from the DF. The mincing procedure was improved, and mincing repeated until the DF appeared pulpy to facilitate the release of single cells. Obtaining the maximum number of single cells increa...

Materiały

Name	Company	Catalog Number	Comments
EMSURE ACS,ISO,Reag. Ph Eur 2-Propanol	EMD millipore Co., MA, USA	1096341011	1 L
0.05% trypsin-EDTA	Gibco, Grand island, NY, USA	25300054	100 mL
40 μm cell strainer	Falcon, NC, USA	352340
Cell culture dish (100 x 20 mm)	Thermo Fisher Scientific Inc., Waltham, USA	172958	Discontinued
Cell culture dish (60 x 15 mm)	Thermo Fisher Scientific Inc., Waltham, USA	150326
Collagenase type 1	Gibco, Grand island, NY, USA	17100017	1 g
Combi-514R / Refrigerated large capacity centrifuge	Hanil science industrial Co., LTD., Daejeon, South Korea	CB-514R
Conical tube	SPL Life Sciences Co., Ltd., Gyeonggi-do, South Korea	50015 50050	15 mL 50 mL
Cryogenic vial	Corning Inc., NY, US	430488	2 mL
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich, Missouri, US	D2650-100ml	100 mL
Dispase	Gibco, Grand island, NY, USA	17105041	1 g
Dulbecco's Phosphate-Buffered Saline	Welgene Inc., Gyengsangbuk-do, South Korea	LB 001-02	500 mL
Fetal bovine serum	Gibco, Grand island, NY, USA	16000044	500 mL
Heraeus BB 15 / CO2 incubator	Thermo Fisher Scientific Inc., Waltham, USA	51023121
Keratinocyte serum-free medium	Gibco, Grand island, NY, USA	10724-011	500 mL
MVE CryoSystem 2000	MVE Biological Solutions Co., GA, USA	CryoSystem 2000
Nalgene Mr. Frosty Freezing Container	Thermo Fisher Scientific Inc., Waltham, USA	5100-0001	for 1.2-2 mL CryoVials
Olympus CKX41 / Inverted cell culture microscope	Olympus Life Science, Waltham, Massachusetts	22-00723-01	Discontinued
Penicillin-Streptomycin Strep	Gibco, Grand island, NY, USA	15140122	100 mL (10,000 U/mL)
Pipet aid XP	Drummond scientific Co., PA, USA	HDR-4-000-201
Pipetman Classic P1000	Gilson, Villiers le Bel, France	F123602	100-1000 µL
Refrigerant	Nihon freezer Co. Ltd., Tokyo, Japan	CLN 540U	~-80 °C / Discontinued
Serological pipet	SPL Life Sciences Co., Ltd., Gyeonggi-do, South Korea	91010	10ml
TrypLE Express Enzyme (1x), phenol red	Thermo Fisher Scientific Inc., Waltham, USA	12605010	cell dissociation protease