Isolation, Culture, and Characterization of Primary Salivary Gland Cells

Abstract

Primary cells are an essential tool for in vitro studies and are obtained directly from living tissues or organs. They closely mimic the physiological state and maintain in vivo functions for short periods of time under optimal conditions. Isolation and culture of salivary gland (SG) cells are useful to decipher the various mechanisms involved in salivary gland dysfunction. However, unlike some other primary cell cultures, SG cell cultures from patient‐derived tissues present several challenges. They are difficult to obtain, culture, expand, and characterize due to their sensitive heterogenous cell population and limited expansion potential. In addition, the majority of saliva‐secreting acinar cells fail to maintain a differentiated state ex vivo for long periods, and eventually succumb to an acinar‐to‐ductal metaplasia, losing their secretory phenotype and functions. Herein, we describe two detailed protocols for primary SG cell isolation, culture, and expansion from human (or mouse) salivary tissues using serum‐free culture media. We also describe the growth kinetics of these primary cells along with their immunocytochemical characterization. © 2022 Wiley Periodicals LLC.
Basic Protocol 1 : Preparation of SG single‐cell culture from freshly obtained human or mouse SG tissues.
Basic Protocol 2 : Preparation of SG explant culture from freshly obtained human or mouse SG tissues

Full text

Isolation, Culture, and Characterization
of Primary Salivary Gland Cells
Xinyun Su,1,2 Sangeeth Pillai,1Younan Liu,1and Simon D. Tran1,3
1McGill Craniofacial Tissue Engineering and Stem Cells Laboratory, Faculty of Dental
Medicine and Oral Health Sciences, McGill University, Montreal, Canada
2Guangdong Provincial Key Laboratory of Stomatology, Department of Operative Dentistry
and Endodontics, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou,
China
3Corresponding author: simon.tran@mcgill.ca
Published in the Cell Biology section
Primary cells are an essential tool for in vitro studies and are obtained directly
from living tissues or organs. They closely mimic the physiological state and
maintain in vivo functions for short periods of time under optimal conditions.
Isolation and culture of salivary gland (SG) cells are useful to decipher the
various mechanisms involved in salivary gland dysfunction. However, unlike
some other primary cell cultures, SG cell cultures from patient-derived tissues
present several challenges. They are difcult to obtain, culture, expand, and
characterize due to their sensitive heterogenous cell population and limited
expansion potential. In addition, the majority of saliva-secreting acinar cells
fail to maintain a differentiated state ex vivo for long periods, and eventually
succumb to an acinar-to-ductal metaplasia, losing their secretory phenotype
and functions. Herein, we describe two detailed protocols for primary SG cell
isolation, culture, and expansion from human (or mouse) salivary tissues us-
ing serum-free culture media. We also describe the growth kinetics of these
primary cells along with their immunocytochemical characterization. © 2022
Wiley Periodicals LLC.
Basic Protocol 1: Preparation of SG single-cell culture from freshly obtained
human or mouse SG tissues.
Basic Protocol 2: Preparation of SG explant culture from freshly obtained hu-
man or mouse SG tissues
Keywords: acinar cells rductal cells rexplant culture rprimary epithelial
cells rsalivary glands rsingle-cell culture
How to cite this article:
Su, X., Pillai, S., Liu, Y., & Tran, S. D. (2022). Isolation, culture,
and characterization of primary salivary gland cells. Current
Protocols,2, e479. doi: 10.1002/cpz1.479
INTRODUCTION
Salivary glands (SGs) are indispensable exocrine organs that produce saliva to maintain
homeostasis in the oral cavity. In mammals, there are three pairs of major salivary glands
(parotid, submandibular, and sublingual glands) and hundreds of minor salivary glands
(de Paula et al., 2017). Each of these major and minor SGs consist of acinar cells, my-
oepithelial cells, and ductal cells supported by a complex neural and vascular supply.
Major SGs are responsible for most of the salivary secretion, while minor SGs contribute
to lubricating the mucosa, as they are dispersed throughout the mucosa in palatal, buc-
cal, lingual, and labial tissues (Hand, Padmanabhan, & Field, 1999; Riva et al., 2000).
Current Protocols e479, Volume 2
Published in Wiley Online Library (wileyonlinelibrary.com).
doi: 10.1002/cpz1.479
© 2022 Wiley Periodicals LLC.
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Tab le 1 Sequence of Events
Time Event
Day 0-1 Tissue digestion and single-cell seeding
Day 4-6 Re-seeding the oating cells into new plates (small colonies start forming)
Day 10-17 Colonies of cells start expanding and connecting
Day 17-21 Cell passaging and re-seeding
Saliva secretion is essential for health maintenance, for example, in digestion, lubrication,
taste, and protection against microorganisms (Pedersen, Bardow, Jensen, & Nauntofte,
2002). Reduced saliva secretion inevitably causes complications such as secondary ram-
pant caries, dysphagia, dysgeusia, and inammation of the oral mucosa (Vissink, Burlage,
Spijkervet, Jansma, & Coppes, 2003). As such, a better understanding of salivary glands
at the cellular level can enhance our success in restoring their secretory function and in
regenerating and bioengineering salivary tissues.
The ‘Human Salivary Gland’ (HSG) cell line has been commonly used in SG research
(Shirasuna, Sato, & Miyazaki, 1981). However, as an immortalized cell line, HSG cells
have limited potential for clinical applications (Tran et al., 2006). Additionally, HSG
was reported as being contaminated with HeLa cells (Lin et al., 2018). Given the lack of
availability of stable cell lines for SG disease modeling and in vitro drug testing, scien-
tists have alternatively used primary SG epithelial cells isolated and characterized from
murine salivary glands (Maimets et al., 2016; Pringle, Nanduri, van der Zwaag, van Os, &
Coppes, 2011; Varghese et al., 2019) and human salivary glands (Beucler & Miller, 2019;
Seo et al., 2019; Su et al., 2016; Tran et al., 2005). Herein, we describe two protocols that
are convenient and reproducible to isolate epithelial cells from human salivary glands:
rst, the single-cell culture method (Basic Protocol 1), and second, the explant culture
method. Single-cell culture is the preferred approach when larger tissues are available
from which to isolate cells, for example, a complete major SG. It allows for complete
utilization of glands and obtaining cell-type-specic cultures for various in vitro appli-
cations. Explant cultures on the other hand are efcient for culturing primary cells out of
smaller tissues, like human minor SGs or glands isolated from mice. This allows us to
maximize the number of cells from a small amount of tissue with high viability. The tech-
nique is less time consuming (Table 1) and more cost-efcient, as no additional enzyme
processing or mechanical dissociation is required to digest the tissues, and the culture can
be achieved using simple surgical instruments and culture reagents routinely available in
labs (Agnihotri, Gaur, & Albin, 2020). However, the type of cells released from the ex-
plants usually depends on the tissue site from which it is isolated. Sometimes, failure to
remove the brous capsule from the gland tissue may lead to limited or no cell release
from these small tissue pieces. Based on our experience, to get an increase in the total
output of cells, it is advisable to use a combination of both single-cell and explant culture
techniques, depending on the type and size of glands.
NOTE: All procedures are performed in a Class II biological hazard ow hood.
NOTE: All work with human samples should be covered under the Institutional Research
Ethics Board. The work presented here adheres to the regulations from the McGill Re-
search Ethics Board. All information on patients and the samples obtained are docu-
mented and are kept condential.
NOTE: All solutions and equipment used in the handling and processing of tissue and
cells must be sterile, and proper aseptic techniques should be used. All personnel han-
dling human samples should wear disposable gloves, disposable gowns, and protective
eyewear.
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NOTE: All incubation steps are performed in a humidied 37°C, 5% CO2incubator un-
less otherwise specied.
BASIC
PROTOCOL 1
PREPARATION OF SALIVARY GLAND (SG) SINGLE-CELL CULTURE
FROM FRESHLY OBTAINED HUMAN OR MOUSE SG TISSUES
In this protocol, we describe the use of Liberase enzymes to digest fresh human and
mouse SG tissues to obtain single cells, which can be used for downstream applications
such as radiation response, drug testing, or tissue engineering. This protocol is usually
applied when larger tissues (such as a complete human SGs) or several smaller tissues
(such as SGs from multiple mice) are available to isolate cells from.
The timeline from tissue harvesting to the culture of single cells (Basic Protocol 1) is
shown in Table 1.
Materials
Fresh human or mouse salivary gland tissues
Washing solution: phosphate-buffered saline (PBS), pH 7.4; or calcium-,
magnesium-, and phenol red–free Hank’s Balanced Salt Solution (HBSS; Gibco,
cat. no. 14175-095) with 2% antibiotic-antimycotic (Gibco, cat. no. 15240-062)
If freshly isolated tissues have bleeding spots or coagulated blood clots, add 1000
U heparin (Sandoz, cat no. 02303086) to washing solution (PBS or HBSS)
Digestion buffer: DMEM medium with 35 μg/L Liberase (Roche, cat. no.
05401119001) and 1% (v/v) antibiotic/antimycotic
Sterile 0.9% NaCl solution
Complete medium: Epi Max (Wisent, cat. no. 002-010-CL; https://www.
wisentbioproducts.com/en/ 113-special-media-systems) with 1%
antibiotic/antimycotic
100-mm plastic cell culture dishes
Ruler and electronic scale to calculate size/weight of sample
Ice or ice packs
Individually wrapped sterile pipettes (5 ml, 10 ml, and 25 ml)
15-ml and 50-ml sterile conical tubes
Surgical scissors (curved), scalpels (Fisher scientic, cat no. 36-101-5346),
tweezers
Micropipette and pipette tips (1000 μl)
GentleMACS tubes (Miltenyi Biotec, cat. no. 5150320302)
GentleMACS dissociator (Miltenyi Biotec)
70-μm cell strainers (Fisher Scientic, cat. no. 22363548)
Humidied, 37°C, 5% CO2cell culture incubator
37°C water bath
Additional reagents and equipment for cell culture techniques including counting
cells (see Current Protocols article: Phelan & May, 2015)
Tissue cleaning
1. Transfer the tissues into a pre-weighed 100-mm plastic dish and measure the size of
the gland by placing the dish on a white paper with a ruler (Fig. 1A).
Usually a single submadibular gland (described here) would weigh between 7 and 15 g
depending on the age of the patient and if they have any underlying disease. Sublingual
glands weigh ∼3-5 g.
We have processed two submandibular (left and right) at once and two sublingial glands
(left and right) at once to speed up the tissue digestion process.
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Figure 1 (A) One human submandibular gland (huSMG). (B) The gland is wetted with small vol-
ume of digestion buffer and mechanically cut with scissors into small pieces. (C) The small tissue
pieces are further minced finely using two scissors simultaneously. (D) The tissue is made into
a fine slurry paste. (E) The entire slurry is transferred into a 50-ml conical tube with the diges-
tion buffer (see materials for Basic Protocol 1). (F) After shaking, the sample is transferred into
gentleMACS c-tubes and processed with the gentleMACS dissociator.
2. Weigh the sample in the dish on the same scale and subtract the weight of the dish to
obtain sample weight. In case of mouse tissues, place two submandibular or several
parotid glands obtained from different mice in one 100-mm dish and prepare as
described below for processing. Keep on ice.
3. Once sample details are obtained, begin tissue cleaning by transferring all the tissue
or the whole SG from the 100-mm dish into a 50-ml conical tube. Add PBS or HBSS
with antibiotic-antimycotic (and optionally heparin), and gently shake the tube up
and down manually (using your hands) for about 45 s to wash the whole gland.
Remove the wash buffer and add fresh PBS/HBSS to wash again.
4. Repeat step 3 at least three times or until the washing solution is clear of blood or
oating brous debris.
Tissue preparation
5. Using ne tweezers and scalpels, remove the brous capsule, connective, and fat
tissues along with any cauterized tissue ends from the SG surface, and proceed to
next step.
6. Place the tissues on a 100-mm dish and add 0.3-0.5 ml of digestion buffer (depend-
ing upon tissue size). Use sharp curved scissors to rst cut the tissues into small
pieces (about 5 mm in size; Fig. 1B). Then, further mince these tissue pieces us-
ing two curved scissors to make into a ne slurry paste (residual tissue pieces are
approximately 1 mm or less; Fig. 1C and D).
Since the salivary gland tissues are soft, it is challenging to mince bigger tissue pieces.
Cutting the tissues rst into small pieces (∼5 mm in size) will improve efciency during
the mincing step, which is done using two sharp curved scissors simultaneously to facil-
itate tissue cutting. One scissor can be used if the operator is not comfortable with using
both hands, but it will take more time to obtain the ne slurry paste.
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7. Transfer this slurry using a 1000-μl micropipette tip (cut the pipette tip at ∼1cm
from the tip using a sterile scissor to widen the opening) into a 50-ml conical tube
and add the digestion buffer.
Volume of digestion buffer depends on the weight of the sample, roughly, 35 ml of diges-
tion buffer for 2-3 g of SG tissue (Fig. 1D and E).
8. Mix the sample with the digestion buffer by vigorous shaking for 30 s using a vortex.
9. Transfer the mixture into gentleMACS tubes (∼15 ml per tube) and use the appro-
priate program on the GentleMACS dissociator (C2, D2; 45-60 s) to vigorously mix
the slurry with the enzymes (Fig. 1F).
10. Transfer the sample back from the gentleMACS tubes to a 50-ml tube, or let it remain
in the same gentleMACS tube (depending on if you wish to repeat step 5 for further
digestion) on a plate shaker inside the cell culture incubator with 5% CO2at 37°C
for 1-3 hr.
Incubation time depends upon the size of tissue—more incubation time leads to better
digestion, but decreases the viability of cells.
11. Every 20-30 min, remove the tube from the incubator and shake it manually for
about 20 s (i.e., hold the tube in one hand and pivot the tube upside down and right
side up for 15-20 times).
This action will disperse the tissue pieces and avoid clumping. Also, this manual shaking
does not add bubbles to the solution.
12. Check the tube during and at end of the incubation time for any remaining undigested
tissue pieces. If the solution appears cloudy and completely homogenous (free of
tiny visible tissue pieces), then proceed to the ltration step.
13. Pass the digested tissue suspension through a 70-μm cell strainer and transfer the
owthrough to a new 50-ml conical tube. Centrifuged 5 min at 300 ×g,4°C.
14. Remove the supernatant carefully and leave about 5 ml of remaining supernatant in
the tube (to avoid sucking off any cells close to the pellet).
15. Add 10 ml of sterile 0.9% NaCl solution to the 50-ml tube and gently mix using
a 1000-μl pipette tip to wash the cells. Centrifuge 45 s at 60 ×gand remove the
supernatant. Repeat the washing and centrifugation steps with 10 ml of 0.9% NaCl
until the supernatant is clear and devoid of the pink color from the enzyme solution
as well as debris.
These steps will dilute and remove as much as possible the digestion enzymes (pink in
color) remaining in the 5 ml solution at the end of step 4. In addition, low centrifugation
speeds allow cells to pellet, while the cellular debris, which are lighter, remain in solution
and thus can be eliminated from the culture).
16. Resuspend the cell pellet in 1-2 ml of Epi Max serum-free medium (depending on
cell number), count the cell number (Phelan & May, 2015) , seed at a density of
∼1-2 ×106cells per 100-mm dish, and incubate at 37°C with 5% CO2.
Maintenance of the single-cell culture
Epi Max is a serum-free medium that comes with three supplements (contents are propri-
etary ) to which we only add 1% (v/v) antibiotic-antimycotic solution for all our cultures.
After passaging salivary gland primary cells, sometimes, if they fail to attach (from P0
to P1), we add 5% fetal bovine serum (see Basic Protocol 2 materials list for cat. no.)
to the Epi Max culture medium to promote cell attachment. After 48 hr, once cells are
attached, we replace with ‘serum free’ Epi Max medium.
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Figure 2 Phase-contrast images showing the morphology of salivary gland cells cultured using
Basic Protocol 1. (A) Epithelial cells on Day 7 at passage 0. (B) Epithelial cells on day 10 after
passage 1. Scale bar: 43 μm.
17. At day 0, when cells are seeded, add 4-5 ml culture medium (Epi Max) to the plate
and leave undisturbed for about 48 hr. After 2 days, add an additional 2-3 ml of
medium to the plates and leave undisturbed for the next 2 days.
18. At day 4-6, small, isolated cell colonies start to appear on the culture plate (Fig. 2).
On this day, collect all the medium from the plate and transfer into a 15-ml conical
tube. Next, wash the culture plate gently using a pipette with Ca-, Mg-, and phenol
red–free HBSS or PBS solution to remove any debris, and add 5-6 ml of fresh culture
medium. Replace with fresh medium every 3-4 days.
19. Centrifuge the medium collected in the 15-ml tubes from the plates for 5 min at 300
×g, 4°C. Remove most of the supernatant and keep ∼1 ml in the bottom of the tube
to avoid sucking up the cell pellet. Then, add 5-10 ml of 0.9% NaCl solution and
centrifuge at 45 s at 60 ×g, 4°C, to remove dead cells, debris, and lysed RBCs.
20. Resuspend the remaining cell pellet with 5-6 ml of fresh culture medium (for 1–2 ×
106cells) using a 1000-μl micropipette, and gently mix the cells before plating into
new 100-mm dishes. Repeat steps 17 and 18 for these re-plated cells (oating cells
in the parent plate at passage 0).
21. At day 4-6 for the new plates (which contain oating cells from the parent plates),
new cell colonies start appearing. On this day, discard all the medium with any re-
maining oating cells and debris, and replace with 5-6 ml fresh culture medium.
Subsequently, replace with fresh medium every 3-4 days.
The time taken for the entire process starting from tissue cleaning to cell seeding depends
upon the size and number of glands. For reference, to process an entire left and right
human submandibular gland, it takes 5-6 hr.
For cell passaging, see Basic Protocol 2, steps 14-22
BASIC
PROTOCOL 2
PREPARATION OF SG EXPLANT CULTURE FROM FRESHLY OBTAINED
HUMAN OR MOUSE SG TISSUES
In this protocol, we provide an alternative method to isolate primary cells from fresh
human or mouse samples by cutting the SG tissues into small pieces (∼1-2 mm) and
growing them in tissue culture plates for downstream applications. We also detail the
steps to successfully passage these isolated cells using trypsin-EDTA.
Materials
Mice (6- 8-week-old; e.g., C57BL/6)
70% ethanol in spray bottle
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Phosphate-buffered saline (PBS), pH 7.4; or calcium-, magnesium-, and phenol
red–free Hank’s Balanced Salt Solution (HBSS); Gibco, cat. no. 14175-095)
with 2% antibiotic-antimycotic (Gibco, cat. no. 15240-062)
Complete medium: Epi Max (Wisent, cat. no. 002-010-CL) with 1%
antibiotic/antimycotic
Trypsin-EDTA (1×), 0.25% (Gibco, cat. no. 15050-057)
Neutralizing medium: DMEM with 10% FBS (Gibco, cat. no. 26140-079) and 1%
antibiotic-antimycotic
Surgical instruments: scalpels, dissecting forceps, scissors
Tissue culture plates (100 mm, 60 mm, 6-well, 12-well; depending on your
application/expected number of cells)
Humidied, 37°C, 5% CO2cell culture incubator
Refrigerated centrifuge
96-well culture plates (optional)
Additional reagents and equipment for euthanasia of rodents (see Current Protocols
article: Donovan & Brown, 2006)
NOTE: All procedures involving live animals must be approved by the local animal care
and use committee and follow governmental and institutional regulations.
Isolation of mice salivary glands
1. Sacrice 6–8-week-old mice (e.g., C57BL/6).
Procedures for euthanasia of mice are described in Donovan & Brown (2006).
There is no specic method of mouse sacrice that is recommended. For isolation
of mouse salivary gland cells, it is best to sedate the mouse, then harvest the sali-
vary gland tissue and follow with CO2euthanasia. Details are described by McGill’s
animal SOP (https://www.mcgill.ca/research/les/research/301-_rodent_euthanasia_-
_july_2021_0.pdf ).
Number of mice to be sacriced depends on how many cells you need and the skill of the
researchers. For practice, 1-2 mice would be recommended to isolate the cells. Going
forward, researchers can sacrice mice based on their demands.
2. Spray 70% ethanol on to the head and neck regions of the mice to sterilize the skin
surface.
3. Use sterilized scalpels, dissecting forceps, and scissors to harvest the submandibular
and sublingual glands from mice.
4. Place the glands on a 100-mm dish and cover entirely with PBS (room temperature).
Human SG explant culture method
5. Follow the tissue cleaning (steps 1-4) and preparation steps (step 5) from Basic Pro-
tocol 1.
6. Mince the salivary gland tissues (human or mice) into 1-2 mm (diameter) pieces
with a sharp scalpel (Fig. 3A).
Always keep all the tissue wet with culture medium.
7. Prepare a 100-mm dish with 2 ml complete medium (Epi Max), and rinse the dish
until medium wets the entire surface of the dish.
8. Disperse the small pieces of sample into the pre-wet 100-mm dish (Fig. 3A).
Do not place the pieces at the edge of the dish. Keep enough distance between the tissue
pieces to provide enough area for the cells to expand.
9. Incubate the dish in the incubator overnight at 37°C with 5% CO2.Su et al.
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Figure 3 Explant culture of human salivary gland cells. (A) Small pieces (1-2 mm) of explant
tissue placed on a 100-mm dish. (B) Piece of minor labial gland attached to culture dish for 14
days; primary salivary cells have migrated to the periphery of the explant culture. (C) Example of
an explant culture from a human submandibular gland (day 18) and (D) of a human parotid gland
(day 21). Scale bar: 90 μm.
Figure 4 Primary SG cell growth kinetics measured using WST-8 cell metabolic assay. Primary
huSMG cells were seeded at a concentration of 2000 cells/well in a 96-well plate at day 0. Samples
were taken at days 0, 1, 3, 5, and 7, and absorbance was measured at 460 nm using a microplate
reader.Primary cells need 24 hr to attach to the culture plates.Once attached, there is a log phase
where the cell metabolism increases, which continues until day 3, suggesting cell proliferation.
After day 3, cells start showing a decrease in metabolic activity, which could be associated with
cell crowding and overlapping. (Standard curve is shown on the left).
10. For the rst 48 hr, keep the culture medium at 2-3 ml per 100-mm dish.
Medium should not cover the whole tissue; otherwise the tissue will fail to attach and
oat. However, if there is an insufcient volume of medium, the tissue pieces will dry and
become necrotic.
11. If needed, add an additional 1-2 ml medium during these 48-72 hr to keep the nal
volume around 3-5 ml in the 100-mm dish.
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Figure 5 (A) E-cadherin, salivary epithelial cell surface marker. (B) Aquaporin-5 (AQP-5), wa-
ter channel protein found in salivary acinar cell membrane. (C) Pan-cytokeratins (wide spectrum
cytokeratin) detect a wide range of cytokeratin filament proteins found in salivary gland epithelial,
and myoepithelial cells. (D) Zona occludens-1 (ZO-1), tight junction–associated protein found in
salivary cell membrane. Scale bar: 70 μm.
12. Change the medium every 2-3 days. Keep a constant check on the medium during
the rst 3-7 days to prevent the tissues from getting dry. When required, add fresh
complete medium gently from the side of the dish to prevent any tissue dislodge-
ment.
13. After 5-7 days, cells begin to grow out from the tissue piece (Fig. 3). During the
rst week, do not move the dish too frequently. When changing and adding culture
medium, move the dish gently. When cells begin to grow from most of the tissue,
change medium every 3 days and replace with 5 ml complete Epi Max medium.
Cell passaging (for Basic Protocols 1 and 2)
14. Timing for cell passage. Passage cells when one of these situations is observed:
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b. Two cell colonies merge to form larger colonies, or
c. Some of the cells begin to die and oat due to cell crowding within an individual
large cell colony/island, which usually takes around 3 weeks.
15. For the explant culture method (Basic Protocol 2), transfer the tissue pieces to a
new pre-wet dish using tweezers. Cells can continue to grow and migrate from these
pieces of tissues again by following the Basic Protocol 2, steps 1-9.
While re-plating these tissue pieces into new dishes, care should be taken with the tips of
the tweezer to avoid damaging the cells released around the tissue explant.
16. Aspirate culture medium in the cell culture dish.
From here, the steps for passaging cells are the same for both Basic Protocols 1 and 2.
17. Wash the dish with 5 ml PBS or HBSS. Aspirate the PBS/HBSS.
18. Add 3 ml 0.25% trypsin-EDTA (cell-detaching solution) to the 100-mm dish. Place
culture dish in a 37°C incubator for 3-5 min. Gently tap the sides of the dishes to
mechanically detach cells.
19. Add 3 ml of neutralizing solution (DMEM with 5%-10% FBS and 1% antibiotic-
antimycotic) to inhibit the trypsin activity. Detach cells by ushing the trypsin +
DMEM (6 ml) with a pipette.
Due to the adherent nature of salivary epithelial cells, the rst trypsinization might only
remove portions of the cell colonies within the 100-mm dish. When this happens, repeat
steps 5 and 6 again to detach the remaining adherent cells.
20. Collect all the detached cells (cells +trypsin +DMEM) into a 15- or 50-ml conical
tube. Centrifuge 5 min at 300 ×g,4°C.
21. Remove the supernatant and resuspend the cell pellet in the conical tube with com-
plete medium (Epi Max with 1% antibiotic-antimycotic). Gently pipette up and
down to obtain a single-cell suspension.
22. Transfer cell solution into two new 100-mm dishes (a 1:2 split ratio).
23. Optional: Cells can also be seeded at a concentration of 2000 cells/well in a 96-well
plate to measure cell proliferation (samples taken at 0, 1, 3, 5, and 7 days; Fig. 4)
or subjected to immunocytochemistry to look at the development of cell markers
(Fig. 5).
COMMENTARY
Background Information
Permanent loss of saliva-secreting acinar
cells due to radiation, Sjögren’s syndrome, or
aging, resulting in hyposalivation and xerosto-
mia, has a substantial negative impact on the
oral health and quality of life of patients. Cur-
rent treatment strategies using lifelong substi-
tutes or drugs to alleviate dry mouth symp-
toms are temporary approaches. An important
step in the development pipeline of treatments
aiming at SG regeneration is designing func-
tional ex vivo human models to study the ef-
fects of drugs, as well as cell and gene thera-
pies, on SGs. Cultured primary SG cells play
a crucial role in developing various in vitro
and ex vivo disease models, as these cells are
able to retain their native SG cell morphology
and thereby the localization of key proteins.
We have previously demonstrated the abil-
ity to culture polarized human primary sub-
mandibular cells with characteristics such as
proper localization of tight junction proteins,
ion channels, and co-transporters for para-
cellular uid movements (Tran et al., 2005).
Using analogous cell culture approaches, we
have developed additional in vitro and ex vivo
SG models over the years for SG researchers
(Maria, Liu, El-Hakim, Zeitouni, & Tran,
2017; Maria & Tran, 2011; Maria, Zeitouni,
Gologan, & Tran, 2011; Seo et al., 2019; Su
et al., 2020). Also during the past decade, we
have tested several culture conditions includ-
ing the use of serum-containing, serum-free,
and dened media to nd an optimal growth
condition for culturing salivary epithelial
cells (Charbonneau, Kinsella, & Tran, 2019;
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Charbonneau & Tran, 2020; Zhang, Pham,
Munguia-Lopez, Kinsella, & Tran, 2020). In
this protocol, we have described a simple and
reproducible method to obtain epithelial cells
from human SG tissues for different down-
stream applications. Our described protocols
have been previously used to culture monkey
salivary epithelial cells as well (Tran et al.,
2006).
Critical Parameters
Using this protocol, we have been able to
successfully isolate, culture, and expand dif-
ferent salivary gland cell types in vitro from
patient samples. Time is very crucial for an
optimal yield of cells after single-cell diges-
tion. Samples should be processed as soon as
they arrive at the lab from the hospital. All tis-
sue cleaning and cutting procedures, until in-
cubation, should be done on ice. Longer pro-
cessing and digestion time leads to lower cell
viability. However, the time needed for each
gland usually depends on the size and qual-
ity of the tissue obtained. Larger salivary tis-
sue with highly brous connective tissue takes
more time to prepare (to make the tissue free
of fat and connective tissues) and digest (more
tissue, longer incubation period). The entire
process must be optimized on the day of re-
ceipt of the glands, and the reagents and arma-
mentarium should be made ready a day before
to avoid delays on the day of processing.
Troubleshooting
Removing the fat and connective tissue is
crucial before cutting the tissues, as their im-
proper removal might necessitate longer incu-
bation time for cell digestion. After cell di-
gestion, ltration with a 70-μm strainer might
pose a challenge, as the strainers tend to clog
after 2-4 ml of the cell solution is passed.
In such situations, use 0.9% NaCl solution to
wash down some of the clogged cells by force
of liquid ow using a pipette or a 10-ml sy-
ringe. If the cell solution still fails to pass
through, change the lter immediately and use
a new lter. After ltration, make sure to wash
the cell pellet multiple times with 0.9% NaCl
to remove as much debris as possible. This will
lead to cleaner cultures when cells are seeded.
When seeding, cells tend to be in clumps. Gen-
tly pipetting the solution up and down will dis-
perse these cell clumps and result in a more
uniform distribution of cells throughout the
culture dish.
Understanding Results
The total yield of cells from Basic Proto-
cols 1 and 2 depends on the quality of tissue.
Glands harvested from older patients have less
lobular gland structure and more brotic tis-
sues, resulting in poor yield. As a reference,
a whole submandibular gland from a 37-year-
old patient can yield up to 25-30 ×106cells in
single-cell culture after processing. For larger
tissue samples, Basic Protocol 1 is preferred,
to make use of the entire gland. Alternatively,
the explant culture method (Basic Protocol 2)
could be used in conjunction with single-cell
digestion to increase total yields. Yields from
single cells are also dependent on how oat-
ing cells attach and expand after replating into
new dishes (see Basic Protocol 1, steps 17-
21). These oating cells, if carefully isolated
and replated in a timely fashion, can maxi-
mize the total number of cells obtained from
the single-cell digestion protocol. These cells
form colonies similar to the parent plate and
retain all the morphological characteristics of
primary SG epithelial cells. In addition, these
replated cell cultures are cleaner due to ab-
sence of any lysed RBCs or cell debris, which
are commonly seen in the parent plates after
initial seeding. For the explant culture method,
when cells are ready to be passaged, the small
pieces of tissue can be removed from the orig-
inal dish with a tweezer and transferred into
a new 100-mm dish pre-wet with medium. We
have successfully continued to grow cells from
these pieces of tissue up to three times.
Time Considerations
Preparation of SG single-cell culture (Basic
Protocol 1) usually takes about 5-6 hr depend-
ing on the size of the tissue sample. The ex-
plant culture (Basic Protocol 2) usually takes 1
hr for tissue washing and cleaning and around
1 hr to plate the small tissue pieces into 100-
mm dishes. For both single-cell and explant
culture (Basic Protocols 1 and 2), it takes
around 17-21 days for the cells to be ready.
The total duration of the primary cell culture
may be about 6-8 weeks, depending on the
number of passages the cells can survive (usu-
ally 2-3 passages).
Acknowledgments
This work was supported in part by
the Canadian Institutes of Health Research
(CIHR). We thank all the anonymous patients
for their salivary gland tissues that were used
in this work. We thank all current and past
members of our lab at McGill University who
contributed toward the development of this
protocol over the years. We also thank all
our collaborating surgeons from McGill Uni-
versity Health Center and Montreal General Su et al.
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Hospital for providing us the human salivary
gland samples.
Author Contributions
Xinyun Su: conceptualization, data cura-
tion, formal analysis, investigation, methodol-
ogy, project administration, validation, visual-
ization, writing original draft, writing review
and editing; Sangeeth Pillai: data curation,
formal analysis, investigation, methodology,
project administration, validation, visualiza-
tion, writing original draft, writing review and
editing; Younan Liu: data curation, formal
analysis, investigation, methodology, project
administration, resources, validation, visual-
ization, writing review and editing; Simon
D. Tran: conceptualization, data curation,
formal analysis, funding acquisition, investi-
gation, methodology, project administration,
resources, supervision, validation, visualiza-
tion, writing original draft, writing review and
editing.
Conict of Interest
The authors have no conicts to declare.
Data Availability Statement
Data openly available in a public repository
that issues datasets with DOIs.
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