In vitro quantification of pigment production and transfer in 2D co-cultures and 3D skin organotypic

Summary

Here, we present a protocol to set up and study 2D keratinocyte-melanocyte co-cultures and 3D full-thickness human skin equivalents. We describe steps for culturing of keratinocyte and melanocyte lines and the establishment of both 2D and 3D co-cultures. The cultures are utilized to measure melanin content and investigate mechanisms driving melanin production and transfer, through flow cytometry and immunohistochemistry. Culture conditions are highly amendable to different conditions, and analysis is simple and objective—thus allowing for medium to high throughput.

For complete details on the use and execution of this protocol, please refer to Ng et al. (2022).¹

Graphical abstract

Highlights

• •Two-dimensional keratinocyte-melanocyte co-cultures
• •Three-dimensional full-thickness skin organotypic includes fibroblasts, keratinocytes, and melanocytes
• •Quantification of pigment production by melanocytes
• •Quantification of pigment transferred to keratinocytes

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol to set up and study 2D keratinocyte-melanocyte co-cultures and 3D full-thickness human skin equivalents. We describe steps for culturing of keratinocyte and melanocyte lines and the establishment of both 2D and 3D co-cultures. The cultures are utilized to measure melanin content and investigate mechanisms driving melanin production and transfer, through flow cytometry and immunohistochemistry. Culture conditions are highly amendable to different conditions, and analysis is simple and objective—thus allowing for medium to high throughput.

Before you begin

Originating from the neural crest cells (NCC), melanocytes are pigment-producing cells present in the epidermis and hair follicles. The life cycle comprises several steps, causing lineage specifications starting from the neural crest cells, migration, proliferation of melanoblasts, proliferation and differentiation into melanocytes, to the maturation of melanocytes, producing melanosomes.² Melanocytes can be found in the basal layer of the epidermis, in an estimated ratio of 10 keratinocytes to 1 melanocyte. That being said, a single melanocyte can be associated with 30–40 keratinocytes in the epidermis through the formation of an epidermal melanin unit.^2,3

A closed paracrine system regulates the transfer of melanin from melanocytes into keratinocytes. Interactions are formed through epidermal melanin units.^3,4,5

Melanosomes packed with melanin are transferred from melanocytes to keratinocytes in globules enclosed by the plasma membrane. These globules are released through the dendrites of melanocytes and phagocytosed by keratinocytes, where they are then dispersed around the perinuclear area.⁶ Contact adhesion between the dendrites of differentiated melanocytes and keratinocytes is imperative for transferring melanin from melanocytes to keratinocytes. The degree of melanin transferred determines the skin color and the degree of photoprotection.² However, the mechanism by which the transfer of melanin occurs is still unclear.

This protocol describes the in vitro approach to mimic the environment for melanin production and transfer in the skin using 2D and 3D methods. The method below describes the specific steps for this protocol using keratinocytes (n/TERT) cells,⁷ and primary melanocytes (HEMn) cells. We have used this protocol to generate skin tone-specific co-cultures using three commercially available melanocyte cell lines – lightly pigmented (HEMn-LP), moderately pigmented (HEMn-MP), and darkly pigmented (HEMn-DP).

Institutional permissions (if applicable)

None.

Preparation of cell culture lines

Timing: 1 day

• 1.
  Culturing of keratinocyte and fibroblast lines.

  • a.
    The keratinocyte and fibroblast lines used in this protocol are cultured using Corning 100 mm TC-treated culture dishes.
  • b.
    Before subculturing or the start of experiment, always view the culture under the microscope to ensure confluency. Culture is ready for subculture when confluency reaches 60%–70%.
  • c.
    To dislodge cells from culture plate, add 2 mL oif Trypsin-EDTA (0.25%), and incubate in 37°C, 5% CO₂ for no longer than 5 min.
  • d.
    To stop trypsin digestion, quench activity by adding 2 mL of 10% HyCylone Defined Fetal Bovine Serum (FBS) in HyClone Dulbecco’s Phosphate Buffered Saline: Liquid (DPBS).
  • e.
    Centrifuge for 3 min at 1,200 rpm.
  • f.
    Passage the cells at a density of 1:20, and incubate in 37°C, 5% CO_2.
• 2.
  Culturing of primary melanocyte lines.

  • a.
    The primary melanocyte lines used in this protocol are cultured using tissue culture plates.
  • b.
    Before subculturing or the start of experiment, always view the culture under the microscope to ensure confluency. Culture is ready for subculture when confluency reaches 70%–80%.
  • c.
    To dislodge cells from culture plate, use 0.05% Trypsin/ EDTA solution, and incubate in 37°C, 5% CO₂ for no longer than 5 min.
  • d.
    To stop trypsin digestion, quench activity by adding 2 mL of 10% FBS in DPBS.
  • e.
    Centrifuge for 3 min at 1,200 rpm.
  • f.
    Passage the cells at a density of 1:10, and incubate in 37°C, 5% CO_2.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Recombinant Anti-Cytokeratin 14 antibody [EPR17350] - Cytoskeleton Marker (ab181595) Dilution: 1:700	Abcam, UK	ab181595
Cytokeratin 10 Monoclonal Antibody (DE-K10) Dilution: 1:200	Thermo Fisher Scientific, USA	# MA5-13705
Anti-Melanoma Associated Antigen 100+ / 7 kDa antibody [NKI/beteb] (ab34165) Dilution: 1:100	Abcam, UK	ab34165
Anti-TRP1 Antibody (G-9): sc-166857 Dilution: 1:100	Santa Cruz Biotechnology, USA	sc-166857
Human CD117/c-kit Antibody Dilution: 1:100	R&D Systems, USA	AF332
Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150073) Dilution: 1:1000	Abcam, UK	ab150073
Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 594) (ab150076) Dilution: 1:1000	Abcam, UK	ab150076
Donkey Anti-Mouse IgG H&L (Alexa Fluor® 647) (ab150107) Dilution: 1:1000	Abcam, UK	ab150107
Donkey Anti-Goat IgG H&L (Alexa Fluor® 647) (ab150131) Dilution: 1:1000	Abcam, UK	ab150131
Recombinant Anti-Melanoma gp100 antibody [EP4863(2)] Dilution: 1:100	Abcam, UK	ab137079
Chemicals, peptides, and recombinant proteins
Sodium hydroxide	Cica-Web, Kanto Chemical Co., Inc, Japan	37184–00
Paraformaldehyde, powder, 95%	Sigma-Aldrich, USA	158127-500G
Hematoxylin solution, Harris	Sigma-Aldrich, USA	HHS16-500ML
Eosin Y solution, aqueous	Sigma-Aldrich, USA	HT110232-1L
Gelatin solution bioreagent	Sigma-Aldrich, USA	G1393-100ML
10× Phosphate buffered saline (PBS), biotechnology grade, 1L	1ST BASE/ Axil Scientific, Singapore	BUF-2041-10×1L
Dulbecco’s Phosphate-Buffered Saline (DPBS), 1×, w/o Ca & Mg, 500mL	Thermo Fisher Scientific, USA	SH30028.02
Kertinocyte SFM, Combo	Thermo Fisher Scientific, USA	17005042
Medium 254 500ML	Thermo Fisher Scientific, USA	M254500
HMGS−2 PMA−FREE 5ML	Thermo Fisher Scientific, USA	S0165
Trypsin-EDTA (0.25%), phenol red, 100 mL ∗VS.∗	Thermo Fisher Scientific, USA	25200056
Trypsin-EDTA (0.05%), phenol red, 100 mL ∗VS.∗	Thermo Fisher Scientific, USA	25300054
Triton™ X-100	Merck, Germany	T8787-100ML
SDS solution, molecular biology grade (10% w/v)	Promega, USA	V6551
CnT-PRIME Epithelial Culture Medium	CELLnTEC, Switzerland	CnT-PR
DMEM, high glucose	Thermo Fisher Scientific, USA	11965092
cOmplete™ Lysis-M	Merck, Germany	04719956001
cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail	Merck, Germany	04693159001
Corning Collagen I, Rat Tail, 100 mg	Corning Inc, USA	354236
Target retrieval solution, pH 9 (10×)	Agilent Technologies, USA	S236784-2
Prolong Diamond Antifade Mount	Thermo Fisher Scientific, USA	P36970
Experimental models: Cell lines
Human Epidermal Melanocytes, neonatal, moderately pigmented donor, (HEMn-MP)	Thermo Fisher Scientific, USA	C1025C
Human Keratinocytes N/TERT -1	Brigham and Women’s Hospital, Department of Dermatology James G. Rheinwald lab.	CVCL_CW92
hTERT-immortalized human normal dermal fibroblast	Gift from Dr Oliver Dreesen’s lab	Gift from Dr Oliver Dreesen’s lab
Software and algorithms
ImageJ	Schneider et al.	https://imagej.nih.gov/ij/
Other
Microplate, 96 well, PS, F-Bottom, Clear	Greiner Bio-One, Austria	655101
ThinCert Cell Culture Insert for 12-well plates, TC, sterile, translucent membrane (PET), pore diameter: 0,4 μm, 4 multiwell plates/box, 48 ThinCert inserts/box	Greiner Bio-One, Austria	665640
ThinCert plate, 12 well, PS, lid, sterile, single packed	Greiner Bio-One, Austria	665110
SEFAR Lab Pak (NITEX)	Sefar AG, Switzerland	03-70/33

Materials and equipment

Synthetic melanin serial dilution

Standard	Final concentration/ μg/mL	Volume of melanin/ μL	Volume of 1 M NaOH /μL
A	100	100 from stock∗	900
B	75	600 from A	200
C	50	400 from B	200
D	25	250 from C	250
E	5	60 from D	240
F	0	0	130

∗When not in use, the synthetic melanin stock (100 μg/mL) should be stored at 4°C. Dispose of reagents after one month.

Permeabilisation Buffer

Reagent	Final concentration	Amount/mL
Gelatin Solution Bioreagent (2%)	0.2%	5
Triton™ X-100	0.1%	0.05
SDS Solution, Molecular Biology Grade (10% w/v)	0.1%	0.5
10× Phosphate Buffered Saline (PBS), Biotechnology Grade, 1L	1×	5
ddH2O	N/A	39.45
Total	N/A	50

• •Blocking Buffer: add 5 mL of 2% Gelatin Solution and 5mL of 10× PBS in 40 mL ddH₂O.
• •Lysis Buffer for Melanin Assay: add 40 mL of 1× PI in 960 mL of Lysis M.
• •3D Co-culture Condition Media: add 1 mL M254 + HMGS-2 PMA Free in 10 mL Keratinocyte-SFM (1×), with L-glutamine, without calcium chloride.
• •Differentation Media: add 30 mL CnT Prime in 20 mL DMEM.

Step-by-step method details

2D in vitro co-culture

Timing: 2 days

To study the accumulation of melanin in keratinocytes after pigment transfer melanocytes, 2D co-culture was established. After which, live melanocytes and keratinocytes will be isolated through FACS. This is done using the cell surface marker, c-Kit. The content of melanin can be quantified through melanin assay.

• 1.
  Trypsinize and count keratinocytes and melanocytes

  Note: Prior to starting this step, do have a semi-confluent dish of keratinocytes and confluent dish of melanocytes accordingly. Alternatives to culture dishes would include culture flasks.

  • a.
    Add Trypsin to both dishes (0.25% Trypsin for keratinocytes, and 0.05% Trypsin for melanocytes).
  • b.
    Incubate for 5 min at 37°C, 5% CO₂.
  • c.
    Neutralize with 10% FBS in PBS (1:1 ratio). Collect in a 15 mL Falcon tube.
  • d.
    Centrifuge at 1200 rpm for 5 min.
  • e.
    Remove supernatant using vacuum aspirator.
  • f.
    Add 1mL of respective cell medias and mix thoroughly.
  • g.
    Count the number of cells.
• 2.
  Plating of co-culture

  Note: On a 2D plane, the physiological ratio between keratinocytes to melanocytes on the basal layer is 10:1. Thus, to mimic the physiological ratio of the epidermis, the ratio for co-culture would also be 10 keratinocytes to 1 melanocyte (Figure 1).

  • a.
    To seed at maximum density, there should be approximately 4 × 10⁴ cells per growth area, cm². The number of cells seeded will correspond to the number of keratinocytes seeded.

    • i.
      For example, the approximate growth area of a 6 cm dish is 21 cm². Thus, the number of keratinocytes to seed would be 8.4 × 10⁵ and the number of melanocytes to seed would be 8.4 × 10⁴.
  • b.
    Add the required volume to achieve the number of both population of cells needed in a 15 mL Falcon tube.
  • c.
    Centrifuge at 1200 rpm for 5 min.
  • d.
    Remove supernatant using vacuum aspirator.
  • e.
    Add 1 mL of melanocyte media, mix well and seed on respective dish.
  • f.
    Top up with melanocyte media.
  • g.
    Incubate for 18–24 h at 37°C, 5% CO₂.

    Optional: Immunofluorescent staining can be done if required. In our case, we have used the K14 and GP100 antibody to label the keratinocytes and melanocytes, respectively.

    Optional: FACs Analysis can be done if required. In our case, we have used the K14 and GP100 antibody to label the keratinocytes and melanosomes, respectively. In this case, cells which are double positive for K14 and GP100 would represent the population of keratinocytes which have taken up melanin.

• 3.
  FACs Analysis.

  • a.
    Trypsinize cells from dishes.

    • i.
      Add 0.25% Trypsin to dishes.
    • ii.
      Incubate for 5 min at 37°C, 5% CO₂.
    • iii.
      Neutralize with 10% FBS in PBS (1:1 ratio). Collect in 5mL round bottom polystyrene tubes.
    • iv.
      Centrifuge at 1200 rpm for 5 min.
    • v.
      Remove supernatant using vacuum aspirator.
  • b.
    Fixation of cells.

    • i.
      Add 4% PFA to tubes, and incubate for 15 min.
    • ii.
      Wash in 2 mL 1 × PBS, then centrifuge at 1200 rpm for 5 min.
    • iii.
      Repeat (ii) once.
  • c.
    Cell permeabilization.

    • i.
      Add 1 mL of permeabilization buffer (0.2% gelatin, 0.1% Triton X, 0.1% SDS) and incubate for 15 min.
    • ii.
      Wash in 2 mL 1 × PBS, then centrifuge at 1200 rpm for 5 min.
    • iii.
      Repeat (ii) once.
  • d.
    Staining of cells with primary antibody mix.

    • i.
      Add primary antibody mix of 1:100 K14 antibody and 1:100 GP100 antibody in 1 × PBS.
    • ii.
      Incubate for 1 h with occasional shaking.
    • iii.
      Wash in 2 mL 1 × PBS, then centrifuge at 1200 rpm for 5 min.
    • iv.
      Discard supernatant.
    • v.
      Add secondary antibody mix of 1:1000 Donkey Anti-Rabbit IgG H&L (Alexa Fluor ® 488) and 1:1000 Donkey Anti-Mouse IgG H&L (Alexa Fluor ® 594) in 1 × PBS.
    • vi.
      Incubate for 40 min with occasional shaking.
    • vii.
      Wash in 2 mL 1 × PBS, then centrifuge at 1200 rpm for 5 min.
    • viii.
      Discard supernatant.
    • ix.
      Add 800 μL of 1 × PBS and mix well.
    • x.
      Add Hoechst 33342 in a ratio of 1:1000 into the tube.
    • xi.
      Filter through filter paper remove small clumps of cells which might potentially clog the machine.
    • xii.
      Proceed for FACs analysis against APC-A and Alexa Fluor 488-A. The single positive population for APC-A represents melanocytes, and single positive population for Alexa Fluor 488-A represents keratinocytes. The double positive population of both APC-A and Alexa Fluor 488-A will represent the population of keratinocytes which have taken up melanin.
    • xiii.
      Analysis of fold difference can be done against the median intensities obtained from the batch analysis.

Note: If you are carrying out your experiment with a similar gating as mentioned in Figure 2, with any other treatment conditions, fold difference analysis can be done by taking the APC-A Mean value of the treatment condition of the gate against that of the control of the same gate. To look at melanin production in melanocytes, this would refer to the gate P1 (Figure 2). To look at melanin transfer from melanocytes into keratinocytes, this would refer to gate P2 (Figure 2).

• 4.
  Live Sorting.

  • a.
    Trypsinize co-cultured cells from dishes.

    • i.
      Add 0.25% Trypsin to dishes.
    • ii.
      Incubate for 5 min at 37°C, 5% CO₂.
    • iii.
      Neutralize with 10% FBS in PBS (1:1 ratio). Collect in 5mL round bottom polypropylene tubes.
    • iv.
      Centrifuge at 1200 rpm for 5 min.
    • v.
      Remove supernatant using vacuum aspirator.
  • b.
    Staining of co-cultured cells using c-Kit.

    Note: In this co-culture of keratinocytes and melanocytes, only melanocytes express the transmembrane tyrosinase kinase receptor (CD117).

    • i.
      Add primary antibody mix of 1:100 c-Kit antibody in 10% FBS in PBS.
    • ii.
      Incubate on ice for 1 h with occasional shaking.
    • iii.
      Wash in 2 mL 10% FBS in PBS, then centrifuge at 1200 rpm for 5 min.
    • iv.
      Discard supernatant.
    • v.
      Add secondary antibody mix of 1:1000 Donkey Anti-Mouse IgG H&L (Alexa Fluor® 647) antibody in 10% FBS in PBS.
    • vi.
      Incubate on ice for 40 min with occasional shaking.
    • vii.
      Wash in 2 mL 10% FBS in PBS, then centrifuge at 1200 rpm for 5 min.
    • viii.
      Discard supernatant.
    • ix.
      Add 800 μL of 10% FBS in PBS and mix well.
    • x.
      Add Hoechst 33342 in a ratio of 1:1000 into the tube.
    • xi.
      Filter through filter paper.
    • xii.
      Proceed for live sorting against [640] 670/30 - APC and collect cells positive and negative in c-Kit staining in separate tubes. Collect 2 × 10⁵ c-Kit- cells and 2 × 10⁴ c-Kit+ cells.
• 5.
  Melanin Assay.

  • a.
    Melanin extraction from collected cells.

    • i.
      Centrifuge collection tubes at 1200 rpm for 5 min.
    • ii.
      Discard supernatant.
    • iii.
      Add lysis buffer (Lysis M + 1× PI) into respective tubes and mix well. For c-Kit- cells, add 100 μL of lysis buffer. For c-Kit+ cells, add 50 μL of lysis buffer.
    • iv.
      Transfer mix into 1.5 mL Eppendorf tubes.
    • v.
      Centrifuge the cell lysate at 14,000 rpm for 10 min.
    • vi.
      Transfer supernatant into a new Eppendorf tube, and centrifuge at 14,000 rpm for 10 min.
    • vii.
      Discard supernatant.
    • viii.
      Dissolve melanin pellet in 120 μL 1 M NaOH.
    • ix.
      Incubate at 80°C for 1 h.
  • b.
    Preparation of melanin standard serial dilution.

    • i.
      Prepare melanin standards according to the table provided in materials and equipment.
    • ii.
      Incubate at 80°C for 1 h with melanin solution as stated in (a).
  • c.
    Measuring absorbance using Spectrometer.

    • i.
      Transfer in duplicates on a 96 well Grenier clear, flat bottom plate.
    • ii.
      Measure absorbance of melanin at 476 nm.
    • iii.
      Analyze and quantify melanin content in collected cells against standards.

Figure 1.

Expected observation of 2D in vitro co-culture

(A) Keratinocytes and melanocytes co-culture.

(B) Melanocyte monoculture.

(C–D) Cell staining of co-culture with keratinocyte marker K14, and melanosome marker GP100. Counterstained with Hoechst 33342.

Scale bar = 100 μM

Figure 2.

Expected observation of FACs Analysis and Sorting of 2D in vitro co-culture

(A) FACs Analysis of co-culture using K14 and GP100 antibodies. The population P1 represents the melanocytes, which stain positive for GP100 (a melanosome marker) and negative for K14 (a keratinocyte marker). The population P3 represents the keratinocytes, which stain positive for K14 and negative for GP100. The population P2 represents melanin transfer, where keratinocytes have successfully up taken melanin, thus positive for both K14 and GP100.

(B) Example sort plot, the population negative for c-Kit represents the keratinocytes, which do not have the receptor CD117 present on their cell membranes. Meanwhile, the population positive for c-Kit represents the melanocytes, which have the receptor CD117 present on their cell membranes.

3D in vitro co-culture of full thickness epidermal skin equivalent

Timing: 2 weeks

• 6.
  Establishment of 3D in-vitro co-culture.

  • a.
    Day –4: Casting of collagen gel + fibroblast mix.
    Prior to starting this step, do have a confluent dish of fibroblasts, trypsinised and on ice, at a concentration of 1 × 10⁶/mL. Alternatives to culture dishes would include culture flasks.

    CRITICAL: All steps should be done on ice, with cold pipette tips. This is to prevent the collagen from polymerising prematurely.

    • i.
      Aliquot 500 μL of Collagen Type I (Rat Tail) into a cold 1.5 mL Eppendorf tube.

      Note: As batch / lot differences in the concentration of collagen can vary substantially, it might be useful to first determine the desired final collagen concentration.

    • ii.
      Add 12.5 μL of 10 × Dulbecco’s Modified Eagle’s Medium – low glucose. The 10 × DMEM will serve as a pH indicator.
    • iii.
      Neutralize with 1 M NaOH dropwise, until neutral pH is obtained. The solution will turn orange pink.

      Note: Approximately 23 μL of 1 M NaOH is required to neutralize per mL of collagen. Do ensure that the NaOH is well dissolved in in the collagen. If you are using a pH strip, the final pH of this mixture should correspond to pH 7–8.

    • iv.
      To the neutralize collagen, add 1 × 10⁵ of MT fibroblasts, and mix well, being careful not to create too many bubbles.
    • v.
      The mixture was then plated onto membrane support ThinCertsTM cell culture inserts with pore size of 0.4 μM, which were then placed into a 12-well tissue culture plate.
    • vi.
      Incubate at 37°C, 5% CO₂.
    • vii.
      After about 30 min, check that the collagen is completely solidified, and add 1mL of fibroblast media into the 12-well tissue culture plate.
    • viii.
      Incubate overnight at 37°C, 5% CO₂.
  • b.
    Day –3: Adding keratinocyte and melanocyte co-culture.
    Prior to starting this step, do have a semi-confluent dish of keratinocytes and confluent dish of melanocytes accordingly. Alternatives to culture dishes would include culture flasks.

    • i.
      Seed co-culture of 10 keratinocytes to 1 melanocyte like in 2D in vitro co-culture on the hardened collagen gel, suspended in 3D co-culture condition media. For a 12-well insert, the number of cells seeded would correspond to 2 × 10⁵ keratinocytes to 2 × 10⁴ melanocytes.
    • ii.
      At the same time, add 1 mL of 3D co-culture condition media into the 12-well tissue culture plate, replacing the media added the day before.
    • iii.
      Refresh media the next day, with 200 μL of 3D co-culture condition media on the insert, and 1 mL of 3D co-culture condition media into the 12-well tissue culture plate.

      Note: Shrinkage of collagen gel is the result of interaction between fibroblasts contracting in the gel, creating their own matrix. This phenomenon is normal.

  • c.
    Day -1: Changing to differentiation media. 3D co-cultures were switched to differentiation media.
  • d.
    Day 0: Airlift. At this stage, 3D co-cultures are elevated to an air-liquid interface.

    • i.
      Carefully remove media from the 12-well insert, until dry.
    • ii.
      Media should be changed every 2 days, with 4 mL of differentiation media to be added in the lower chamber of the deep well plate

      Note: Throughout the airlift period of 10 days, the 3D co-culture should remain dry to ensure proper stratification of the epidermal layer. At any point of media change, if liquid is observed on the insert with co-culture, gently remove using a pipette.

  • e.
    Day 10: Harvesting of 3D co-culture

    • i.
      Harvest 3D co-culture from insert and fix in 4% paraformaldehyde for 24 h in 4°C.
    • ii.
      Excise in half and embed half in O.C.T. and the other half in cassettes for paraffin processing and embedding. For sectioning, collect 5 μm sections for paraffin sections (FFPE) and 10 μm sections for O.C.T. sections.
• 7.
  Staining of 3D co-culture sections for observation

  • a.
    Hematoxylin & Eosin (H&E) staining is performed to observe the histology of the 3D co-cultures (Figure 3).

    • i.
      The staining is done on FFPE sections in steps shown in the table below.
    • ii.
      Slides are then left to dry and then sealed with coverslips.
    • iii.
      Stained sections were imaged and analyzed with Image J.

      Step	Reagent	Duration
      1	Hematoxylin	2 min
      2	Running water	2 min
      3	Scotts’ bluing water	2 min
      4	Running water	2 min
      5	Eosin Y Aqueuos	20 s
      6	70% Ethanol	1 dip
      7	80% Ethanol	5 s
      8	90% Ethanol	5 s
      9	100% Ethanol	3 min
      10	100% Ethanol	3 min
      11	Xylene	3 min
      12	Xylene	3 min

  • b.
    Immunofluorescent staining is performed to characterize keratinocyte stratification in the 3D co-cultures (Figure 3).

    Note: All incubation steps in this section are done in the dark.

    • i.
      The staining can be done on both FFPE and O.C.T. sections.
    • ii.
      For FFPE, slide sections must go through deparaffinisation and antigen retrieval using 1 × Concentrate of Target Retrieval Solution, and incubated in a pressure cooker.
    • iii.
      Add permeabilisation buffer and incubate for 15 min
    • iv.
      Wash in 1 × PBS twice for 5 min.
    • v.
      Add blocking buffer and incubate for 1 h.
    • vi.
      Add primary antibody mix as showed in the table below. Incubate overnight in 4°C.
    • vii.
      Wash in 1 × PBS twice for 5 min. Repeat this step 3 times.
    • viii.
      Add respective secondary antibody mix as shown in the table below. Incubate for 1 h.
    • ix.
      Wash in 1 × PBS twice for 5 min.
    • x.
      Add Hoechst in ratio 1:1000 in PBS and incubate for 15 min.
    • xi.
      Wash in 1 × PBS twice for 5 min.
    • xii.
      Seal with Prolong Diamond Antifade mounting media.
    • xiii.
      Image under confocal microscope and analyze with Image J.

      Antibodies used for frozen O.C.T. sections
      Primary antibody	Dilution (in 0.2% gelatin / Blocking buffer)	Respective secondary antibody	Dilution (in 1 × PBS)
      Rabbit monoclonal to Anti-Cytokeratin 14	1:700	Donkey Anti-Rabbit IgG Alexa Fluor® 594	1:1000
      Mouse monoclonal to Cytokeratin 10 (DE- K10)	1:200	Donkey Anti-Mouse IgG Alexa Fluor® 647	1:1000
      Mouse monoclonal [NKI/beteb] to Melanoma Associated Antigen 100+/7 kDa	1:100	Donkey Anti-Mouse IgG Alexa Fluor® 488	1:1000
      Antibodies used for FFPE sections
      Rabbit monoclonal to Anti-Cytokeratin 14	1:700	Donkey Anti-Rabbit IgG Alexa Fluor® 488 (Abcam, UK)	1:1000
      Mouse monoclonal to Cytokeratin 10 (DE- K10)	1:200	Donkey Anti-Mouse IgG Alexa Fluor® 594	1:1000
      Mouse monoclonal to TRP 1 (G-9)	1:100	Donkey Anti-Mouse IgG Alexa Fluor® 594	1:1000

• 8.
  Functional quantification of pigment production and transfer regulation.

  Note: This step serves to determine the physiological relevance of findings obtained from 2D in vitro co-cultures. To score the pigment production and transfer, quantification was done by scoring the number of double positive staining of NKI within the basal layer (K14+/NKI+), and in the suprabasal layer (K10+/NKI+).

  • a.
    Quantification was done via an Image J script to encourage consistency of quantification between samples and eliminates possible bias which might occur.
  • b.
    The Image J script used is as follows.
  • c.
    The only manual counting required would be the measuring of the total length of the co-culture organotypic.

    Note: Confocal imaging as stated in this protocol is done using the STELLARIS Confocal Microscope, and thus saved data have the extension .lif. If data was saved with another extension, do alter the code accordingly.

    path = getDirectory("Choose a Directory ");

    outputFolder = getDirectory("Choose_a_Directory ");

    filelist = getFileList(path); //load array of all files inside input directory

    run("Bio-Formats Macro Extensions");

    processBioFormatFiles(path);

    function processBioFormatFiles(path) {

    for (i = 0; i < filelist.length; i++){

    if(endsWith(filelist[i], "lif")){

    Ext.setId(path + filelist[i])'

    Ext.getSeriesCount(seriesCount);

    run("Bio-Formats Importer", "open=["+ path + filelist[i] +"] color_mode=Default rois_import=[ROI manager] view=Hyperstack stack_order=XYCZT series_"+seriesCount);

    name=getTitle();

    run("Duplicate...", "duplicate");

    stack = getImageID(); //original

    close("\\Others");

    selectImage(stack);

    run("Duplicate...", "title=melanosomes duplicate channels=2"); //NKI

    selectImage(stack);

    run("Duplicate...", "title=basal duplicate channels=3"); // basal

    selectImage(stack);

    run("Duplicate...", "title=suprabasal duplicate channels=4"); //suprabasal

    imageCalculator("AND create", "melanosomes", "basal");

    run("Duplicate...", "title=basal_melanin");

    run("Auto Threshold", "method=Yen white");

    setThreshold(1, 255);

    setOption("BlackBackground", true);

    run("Convert to Mask");

    run("Divide...", "value=255");

    run("Measure");

    imageCalculator("AND create", "suprabasal", "basal");

    run("Duplicate...", "title=basal_suprabasal");

    imageCalculator("AND create", "basal_suprabasal", "melanosomes");

    run("Duplicate...", "title=suprabasal_melanin");

    run("Auto Threshold", "method=Yen white");

    setThreshold(1, 255);

    setOption("BlackBackground", true);

    run("Convert to Mask");

    run("Divide...", "value=255");

    run("Measure");

    close("∗"); //closing all images in loop cycle

    }

    }

    }

    saveAs("Results", outputFolder+"results"+".csv");

    close("Results");

Figure 3.

Expected observation of 3D in vitro co-culture

(A–D) (A) Hematoxylin & eosin staining (B–D) Immunofluorescent staining of the 3D in vitro co-culture using K14, K10, TRP and NKI antibodies. All IF staining were counter-stained with Hoechst 33342.

Scale bar = 100 μM

Limitations

Throughout the airlift period of 10 days, the 3D co-culture should remain dry to ensure proper stratification of the epidermal layer. At any point of media change, if liquid is observed on the insert with co-culture, gently remove using a pipette.

It is also imperative to ensure that the CO₂ levels in the tissue culture incubator remains at 5%. This is because any increase of decrease of CO₂ levels might change the pH of the media in the co-culture. This might cause inaccuracies and lapse of consistency in results.

Troubleshooting

Problem 1

In points 3 and 4 in the section: 2D in vitro co-culture, upon viewing co-culture under the microscope to ensure confluency, it is possible to observe many dead cells. There are a few reasons for this.

• •There is a possibility that the cells have been passaged too many times. This is applicable for the primary melanocytes in culture.
• •Another possibility would be that the cells are too confluent. This is a likely issue to experience as cocultures are seeded with high confluence to ensure interaction of keratinocytes and melanocytes – mimicking the basal layer of the epidermis. However, this comes with a caveat that cells are likely way past the log phase and exponential phase of cell growth. This quickly leads to the death phase.

Potential solution

To overcome these possible confounding factors.

• •In our experience, the number of dead cells observed is reduced when melanocytes have been passaged less than a total of 16 doublings. It is also imperative to ensure that cells are not left to sit on the bench without culturing for a long time – as this would lead to increased cell death as well.
• •In our experience, viewing the co-culture and starting our experiments 22–24 h after the initial seeding of co-culture resulted in healthy cultures and a low rate of cell death.

Problem 2

In point 1b in the section: 3D in vitro co-culture of full thickness epidermal skin equivalent, it is possible to observe the collagen gel with numerous air bubbles. There are a few reasons for this.

• •There is a possibility that the collagen mix was not cold enough during its establishment in point 1a.
• •There is another possibility that the pipette tips and tubes used during its establishment in point 1a was too warm – introducing heat into the mix.
• •There is also another possibility of the pipetting movement used in point 1a was too quick, introducing air bubbles.

Potential solution

To overcome these possible confounding factors.

• •
  In our experience, the best way to overcome these confounding factors would include:

  • ○
    Creating the collagen mix on ice throughout its establishment as explained in point 1a.
  • ○
    Pre-cooling of needed equipment by putting them into the fridge before starting the experiment.
  • ○
    Slowing down pipetting movement, as the mix might get viscous while it is cold. It would also be good practice to avoid the second stop on the pipette, as this would usually inevitably introduce unwanted air bubbles.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Carlos Clavel carlos_clavel@asrl.a-star.edu.sg.

Materials availability

This study did not generate new unique reagents.

Data and code availability

The published article includes all [datasets/code] generated or analyzed during this study.