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STAR Protoc
STAR Protoc
STAR Protocols
2666-1667
Elsevier

S2666-1667(24)00429-5
10.1016/j.xpro.2024.103264
103264
Protocol
Protocol to fabricate elastomer microwells for three-dimensional culture of primary adipocytes
Beeghly Garrett F. gfb48@cornell.edu
13∗
Deng Jenny 1
Fischbach Claudia cf99@cornell.edu
124∗∗
1 Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA
2 Kavli Institute at Cornell for Nanoscale Science, Cornell University, Ithaca, NY 14853, USA
∗ Corresponding author gfb48@cornell.edu
∗∗ Corresponding author cf99@cornell.edu
3 Technical contact

4 Lead contact

14 8 2024
20 9 2024
14 8 2024
5 3 103264© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

Our understanding of how adipocytes influence metabolic signaling, immune function, and cancer progression remains limited as the culture of primary adipocytes is challenging. Here, we present a protocol to fabricate elastomer microwells for three-dimensional culture of collagen-embedded adipocytes. We describe steps to cure and functionalize elastomer microwells and to isolate and embed primary adipocytes. We then detail how to culture and analyze adipocyte-collagen gels. This protocol provides broad applications to improve our understanding of adipocyte biology in health and disease.

Graphical abstract

Highlights

• Fabrication and surface functionalization of elastomer microwells

• Isolation of adipocytes from resected human or murine adipose tissue

• Encapsulation of adipocytes in collagen for long-term culture

• Compatible with inhibitor studies, co-culture with other cells, and confocal microscopy

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

Our understanding of how adipocytes influence metabolic signaling, immune function, and cancer progression remains limited as the culture of primary cells is challenging. Here, we present a protocol to fabricate elastomer microwells for three-dimensional culture of collagen-embedded adipocytes. We describe steps to cure and functionalize elastomer microwells and to isolate and embed primary adipocytes. We then detail how to culture and analyze adipocyte-collagen gels. This protocol provides broad applications to improve our understanding of adipocyte biology in health and disease.

Subject areas

Cell Biology
Metabolism
Tissue Engineering
Biotechnology and bioengineering
Material sciences
==== Body
pmcBefore you begin

Adipocytes are the functional unit and most abundant cell type of white adipose tissue. Once considered to be passive energy-storing cells, recent transcriptomic studies reveal distinct subpopulations of adipocytes in humans and mice.1,2 Indeed, adipocytes actively mediate metabolic health,3 immune function,4 and malignant transformation of skin,5 breast,6 and ovarian7 cancers. However, adipocytes are post-mitotic and buoyant in aqueous media, making them difficult to maintain in culture for mechanistic studies. Thus, prior research often relied on preadipocytes that could be seeded and differentiated in vitro. However, these cells represent an early stage of adipogenesis and fail to recapitulate key aspects of mature adipocytes with standard differentiation protocols.8 Current techniques to culture primary adipocytes float cells suspended in culture media9 or immobilize cells beneath tissue-culture inserts10 or within fibrin gels.11 While these approaches enable ex vivo culture of isolated adipocytes, cell viability rapidly decreases in suspension, tissue-culture inserts limit imaging resolution for downstream analysis, and fibrin is not a native component of adipose tissue. Here, we present a protocol to fabricate and functionalize elastomer microwells to enable adhesion and culture of primary adipocytes embedded in collagen type I, which constitutes a major extracellular matrix component of white adipose tissue and influences tissue-resident cell behavior.12,13,14 Moreover, this method enables a high adipocyte-to-collagen volume ratio (three-to-one) to better recapitulate the structure of adipose tissue. Compared to other approaches, this protocol facilitates repeated media exchange, direct or indirect co-culture studies, and high-resolution imaging via confocal microscopy.

Institutional permissions

All research performed with human tissue must conform with the Declaration of Helsinki. Unless samples are collected as de-identified waste with no associated clinical information, all human subjects must provide written informed consent prior to the donation of tissue samples and all associated protocols must be approved in advance by appropriate institutional committees.

All research performed with animal tissue must be approved in advance and performed in accordance with guidelines set by the appropriate institutional committees for animal care and use.

Prepare and cure silicone elastomer

Timing: 5 h

1. In a disposable container, combine 30 g of liquid silicone elastomer base and 3 g of liquid silicone elastomer curing agent from the SYLGARD 184 Silicone Elastomer Kit.

2. Vigorously mix the silicone elastomer solution for 5 min by hand to ensure the components are well combined.

3. Pour 27 g of the resulting elastomer solution into a 150 mm diameter Petri dish. Gently tilt the Petri dish to evenly spread the solution (Figure 1A).Figure 1 Fabrication of elastomer microwells

Elastomer solution before (A) and after (B) degassing with a desiccator. Cured elastomer circles (C) and rings (D and E). Final elastomer microwell after bonding to a glass coverslip (F).

Note: The uncured silicone elastomer solution is extremely viscous and extra solution is prepared to account for volume loss when transferring containers.

4. Use a desiccator and vacuum pump to remove bubbles from the elastomer solution (Figure 1B).a. Remove the Petri dish lid and place the elastomer solution in the desiccator.

b. Seal the desiccator and establish a vacuum.

c. Leave the solution for 5 min to begin removing bubbles.

d. Slowly release the vacuum to pop bubbles at the surface of the elastomer solution.

e. Repeat steps b – d until no bubbles remain.

5. Place the degassed solution in an oven at 60°C for a minimum of 4 h to cure. Alternatively, the solution can cure at 21°C for a minimum of 24 h.

Note: This protocol produces a silicone elastomer that is approximately 1.5 mm thick. Depending on the application, the elastomer thickness can be varied by altering the mass of elastomer solution cured (maintain 10 parts base to 1 part curing agent) or the dimensions of the curing vessel.

Pause point: Cured silicone elastomer can be prepared in advance. Store covered and wrapped in Parafilm to keep the elastomer clean of dust and debris.

Prepare and clean glass coverslips

Timing: 1 h

6. Place 12-mm glass coverslips in a container and submerge them in a solution of 70% (v/v) absolute ethanol in deionized water.

7. Incubate the coverslips on a laboratory rocker or shaker for 30 min at 21°C.

8. Wash the coverslips to remove ethanol and any remaining residue on the glass.a. Aspirate the ethanol solution and replace with deionized water.

b. Incubate the coverslips on a laboratory rocker or shaker for 5 min.

c. Aspirate the deionized water and replace with fresh water.

d. Incubate the coverslips on a laboratory rocker or shaker for 5 min.

e. Repeat steps c – d two additional times.

9. Using forceps, transfer the coverslips to a 150-mm Petri dish and arrange in a single layer.

Note: We recommend using fine, curved forceps to handle the coverslips and lining the Petri dish with filter paper to facilitate removal of the coverslips after drying.

10. Use a Kimwipe to gently blot any excess water from the coverslips.

11. Allow the coverslips to dry completely before proceeding.

Pause point: Cleaned glass coverslips can be prepared in advance. Store covered and wrapped in Parafilm to keep the glass clean of dust and debris.

Assemble elastomer microwells

Timing: 1 h

12. Using a 10-mm biopsy punch, cut out circles of the cured silicone elastomer and transfer to a new Petri dish with forceps (Figure 1C).

13. Using a 6-mm biopsy punch, cut out a smaller hole in the center of each 10-mm circle to form silicone elastomer rings (Figures 1D and 1E).

14. Surface treat the elastomer rings and cleaned glass coverslips using a plasma cleaner.a. Place elastomer rings and coverslips onto a 150-mm Petri dish wrapped in aluminum foil and into the plasma cleaner.

b. Establish a vacuum and treat elastomer rings and coverslips on high for 2 min, starting once the plasma cleaner chamber turns purple.

c. Slowly release the vacuum and remove elastomer rings and coverslips.

d. Using forceps, place an elastomer ring treated-side-down onto the center of a glass coverslip treated-side-up.

e. Using the back of the forceps, gently press down on the surface of the elastomer ring in several locations to facilitate covalent bonding to the coverslip.

f. Repeat steps d – e for all treated elastomer rings and coverslips.

Note: Complete covalent bonding within 15 min of plasma treatment. If needed, perform step 14 in batches and repeat until all microwells are assembled.

15. Transfer each bonded elastomer microwell (Figure 1F) to a well of a 24-well plate for storage.

Pause point: Bonded elastomer microwells can be prepared in advance. Store covered and wrapped in Parafilm to keep the microwells clean of dust and debris for up to 2 weeks.

Surface treat and functionalize elastomer microwells

Timing: 1 h

16. Remove the lid and place the 24-well plate with elastomer microwells into the plasma cleaner; place the lid face-up beside the plate.

17. Establish a vacuum and treat the well plate and lid on high for 5 min, starting once the plasma cleaner chamber turns purple.

18. Slowly release the vacuum and immediately place the lid back on the 24-well plate. Transfer to a biological safety cabinet and maintain aseptic technique from this point onward.

19. Add 40 μL of sterile 1% poly(ethyleneimine) (w/v) in deionized water to the center of each elastomer microwell and incubate for 10 min at 21°C.

20. Aspirate the poly(ethyleneimine), using a sterile pipette tip in your non-dominant hand to hold the microwell in place if needed.

21. Add 40 μL of sterile 0.1% glutaraldehyde (v/v) in deionized water to the center of each elastomer microwell and incubate for 30 min at 21°C.

22. Aspirate the glutaraldehyde and wash each well of the 24-well plate with 1 mL of sterile deionized water; repeat this process two additional times.

23. Store functionalized elastomer microwells in deionized water at 4°C for up to 24 h before use.

CRITICAL: Ensure poly(ethyleneimine) and glutaraldehyde solutions are freshly prepared to functionalize the microwells. Once diluted, glutaraldehyde begins to self-crosslink and should be discarded after 1 month of storage at 4°C. Insufficient fixation of poly(ethyleneimine) to the microwell will result in gel detachment due to the substantial buoyant force exerted by primary adipocytes.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Biological samples	
	
Human adipose tissue	N/A	N/A	
Murine adipose tissue	N/A	N/A	
Collagen type I, high concentration, rat tail	Corning	Cat#354249	
	
Chemicals, peptides, and recombinant proteins	
	
SYLGARD 184 Silicone Elastomer Kit	Ellsworth Adhesives	Cat#4019862	
Poly(ethyleneimine) (PEI) solution	Sigma-Aldrich	Cat#P3143-100ML	
Glutaraldehyde, 50% aqueous solution	Thermo Fisher Scientific	Cat#ICN19859580	
Bovine serum albumin (BSA), fraction V	Thermo Fisher Scientific	Cat#BP1600-100	
Hank’s buffered salt solution (HBSS) 10x, calcium, magnesium, no phenol red	Thermo Fisher Scientific	Cat#14065056	
Gibco Dulbecco’s modified Eagle’s medium (DMEM), low glucose, pyruvate, powder	Thermo Fisher Scientific	Cat#31-600-083	
Ham’s F-12 nutrient mix, powder	Thermo Fisher Scientific	Cat#21700075	
Fetal bovine serum (FBS)	Atlanta Biologicals	Cat#S11150	
Penicillin-streptomycin	Thermo Fisher Scientific	Cat#15070063	
Collagenase type I, 125 units per mg dry weight	Worthington Biochemical	Cat#LS004197	
Hoechst 33342, trihydrochloride trihydrate (2 μg/mL dilution)	Thermo Fisher Scientific	Cat#H3570	
Propidium iodide, 1 mg/mL solution in water (2 μg/mL dilution)	Thermo Fisher Scientific	Cat#P3566	
Paraformaldehyde, granular	Electron Microscopy Sciences	Cat#19208	
4′,6 Diamidino 2 phenylindole, dihydrochloride (DAPI) (2.5 μg/mL dilution)	Thermo Fisher Scientific	Cat#D1306	
Alexa Fluor 488 phalloidin (0.165 μM dilution)	Thermo Fisher Scientific	Cat#A12379	
	
Other	
	
Petri dishes, polystyrene, non-sterile, 150 mm diameter	Neta Scientific	Cat#USS-K1001-PK-180	
Glass coverslips, 12 mm diameter	Chemglass	Cat#CLS-1760-012	
Biopsy punch, 10 mm	Thermo Fisher Scientific	Cat#NC9236770	
Biopsy punch, 6 mm	Integra Miltex	Cat#33-36	
Cell strainers, sterile, 200 μm	pluriSelect	Cat#43-50200-03	
Universal P200 barrier tips, sterile, wide bore	Axygen	Cat#14-222-730	
Universal P1000 barrier tips, sterile, wide bore	Thermo Fisher Scientific	Cat#2079GPK	
Nalgene transparent polycarbonate desiccator	Thermo Fisher Scientific	Cat#5311-0250	
High power expanded plasma cleaner, 115V	Harrick Plasma	Cat#PDC-001-HP	
High vacuum pump	DVP	Cat#RC-4M	
Tube revolver and rotator	VWR	Cat#10054-316	

Materials and equipment

1x Krebs-Ringer HEPES (KRH) Buffer

Reagent	Final concentration	Amount	
Deionized water	N/A	500 mL	
NaCl	116 mM	6.78 g	
HEPES	25 mM	5.96 g	
D-glucose	2 mM	360 mg	
KCl	4 mM	298 mg	
CaCl2	1.8 mM	200 mg	
MgCl2	1 mM	95.2 mg	
Total	N/A	500 mL	
Bovine serum albumin (BSA)	1% (w/v)	5 g	
Adjust pH of 1x KRH buffer between 7.1 – 7.2. Base 1x KRH buffer without BSA can be stored at 21°C for 6 months. Add BSA to the volume of base 1x KRH Buffer needed before use, sterile filter, and store at 4°C for up to 1 week.

1x Hanks Buffered Salt Solution (HBSS)

Reagent	Final concentration	Amount	
Deionized water	N/A	450 mL	
10x HBSS	1x	50 mL	
NaHCO3	2.2 g/L	1.1 g	
Total	N/A	500 mL	
Bovine serum albumin (BSA)	2% (w/v)	10 g	
Adjust pH of 1x HBSS between 7.1 – 7.2. Base 1x HBSS buffer without BSA can be stored at 21°C for 6 months. Add BSA to the volume of base 1x HBSS needed before use and store at 4°C for up to 1 week.

Complete Media

Reagent	Final concentration	Amount	
Deionized water	N/A	445 mL	
DMEM, low glucose, powder	4.99 g/L	2.495 g	
Ham’s F-12 nutrient mix	5.3 g/L	2.65 g	
NaHCO3	2.44 g/L	1.22 g	
Fetal bovine serum (FBS)	10% (v/v)	50 mL	
Penicillin-streptomycin	1% (v/v)	5 mL	
Total	N/A	500 mL	
Adjust pH of complete media between 7.1 – 7.2. Sterile filter and store at 4°C for up to 1 month.

10x Media

Reagent	Final concentration	Amount	
Deionized water	N/A	50 mL	
DMEM, low glucose, powder	49.9 g/L	2.495 g	
Ham’s F-12 nutrient mix	53 g/L	2.65 g	
Total	N/A	50 mL	
Add DMEM and Ham’s F-12 to 40 mL of deionized water and then adjust final volume to 50 mL with additional water. Do not adjust pH. Sterile filter and store at 4°C for up to 6 months.

Collagen Type I Solution

Reagent	Final concentration	Amount	
Collagen type I	2.5 mg/mL	(2.5 mg/mL divided by the stock concentration) multiplied by the total volume	
10x media	N/A	0.1 of total volume	
1N NaOH	N/A	0.023 of collagen type I volume	
Complete media	N/A	Remaining volume	
Total	N/A	Calculated in step 21	
Prepare collagen type I solution as outlined in steps 21–25. Do not prepare in advance.

Step-by-step method details

Isolate primary adipocytes from resected adipose tissue

Timing: 2 h

In this step, human or murine adipose tissue is mechanically dissociated and enzymatically digested with collagenase. The resulting primary adipocytes are separated from stromal vascular cells by buoyancy and repeated washes.1. Obtain resected human or murine adipose tissue in a 50 mL centrifuge tube filled with complete media (Figure 2A). Store tube on ice until use. Maintain aseptic technique from this point onward.Figure 2 Isolation of primary adipocytes

Resected murine adipose tissue before (A) and after (B) mechanical dissociation. Collagenase digested adipose tissue before (C) and after (D) washes to remove stromal vascular cells. Isolated adipocytes before (E) and after (F) packing via centrifugation. Blue arrow denotes 1x KRH buffer. Yellow arrow denotes free lipid. Final adipocyte-collagen gels cast in elastomer microwells (G).

2. In a biological safety cabinet, decant resected adipose tissue into a new 50 mL centrifuge tube along with 10 mL of complete media.

3. Using sterile long-blade scissors, mince the resected adipose tissue for 5 min or until the tissue is separated into pieces a few mm3 in size (Figure 2B).

4. Add 1x KRH buffer to the minced adipose tissue until the 50 mL mark of the centrifuge tube is reached and allow the tissue pieces to rest undisturbed to separate by buoyancy.

5. While the tissue pieces separate, add 1.5 mg/mL of collagenase type I to 1x HBSS pre-warmed to 37°C.

Note: The volume of collagenase type I solution prepared must be greater than or equal to the volume of adipose tissue to achieve sufficient digestion.

6. Sterilize the resulting collagenase type I solution using a 0.22 μm pore filter.

CRITICAL: To maintain enzymatic activity, 1x HBSS is pre-warmed, supplemented with 1.5 mg/mL collagenase type I, and sterile filtered immediately before use. Do not prepare in advance.

7. Decant floating adipose tissue pieces from step 4 into a new 50 mL tube and discard any remaining tissue.

Note: Non-buoyant tissue pieces contain significant amounts of contaminating non-adipose tissue and are removed in this step to improve the purity of isolated adipocytes.

8. Remove as much infranatant as possible beneath the adipose tissue pieces using an aspirator with manual suction control.

Note: If an aspirator with manual suction control is not available, a serological pipette and pipette aid or a blunt needle and syringe can be used to remove the infranatant.

9. Add the pre-warmed collagenase solution to the adipose tissue pieces.

10. Place the tube containing adipose tissue pieces into a tube rotator at 37°C. Incubate until 80%–90% of the tissue pieces are digested or up to a maximum incubation time of 50 min (Figure 2C).

11. Pass the resulting cell suspension through 200 μm cell strainers to remove undigested adipose tissue pieces. Replace as needed if the cell strainers become clogged.

12. Allow the cell suspension to sit undisturbed for 10 min to allow the isolated adipocytes to separate from stromal vascular cells due to buoyancy (Figure 2D).

13. Aspirate the infranatant beneath the layer of floating adipocytes.

Optional: The infranatant can also be reserved to isolate stromal vascular cells for matched co-culture studies. See Seo et al.13 and Ling et al.14 for more details.

14. Add 1x KRH buffer to the adipocytes until the 50 mL mark of the centrifuge tube is reached. Tighten the lid and gently invert the tube several times to wash the isolated adipocytes and remove additional stromal vascular cells.

15. Repeat steps 12–14 two additional times.

Prepare primary adipocytes for embedding

Timing: 15 min

In this step, low-speed centrifugation packs the adipocytes and removes excess 1x KRH buffer and free lipid from the floating adipocyte layer to ensure consistent cell seeding.16. Using wide bore tips and a P200 pipette, transfer the floating adipocyte layer from the top of the 1x KRH buffer and into a microcentrifuge tube (Figure 2E).

Note: Aspirate spillover 1x KRH buffer from the microcentrifuge tube as needed using a blunt needle and syringe.

17. Spin the adipocytes at 21°C for 3 min at 100 × g using a microcentrifuge (Figure 2F).

18. Remove the infranatant beneath the adipocytes with a blunt needle and syringe.

19. Remove any free lipid above the adipocytes with a P200 pipette.

CRITICAL: Failure to remove the infranatant or free lipid will interfere with collagen polymerization and yield inconsistent cell seeding. Ensure both are removed before proceeding with the protocol.

20. Primary adipocytes are now packed and ready for use.

Note: Estimate the final volume of isolated adipocytes and calculate the desired number of elastomer microwells to be prepared. Approximately 30 μL of packed adipocytes are needed per microwell. Refer to problem 1 in troubleshooting if a low yield of adipocytes is obtained.

CRITICAL: Adipocytes are fragile and can rupture due to excessive centrifugation and pipetting. Reduce centrifugation speeds and times to maximize viability when packing adipocytes prior to cell seeding.

Prepare collagen solution and cast adipocyte-collagen gels

Timing: 1 h

In this step, primary adipocytes are embedded in collagen type I for three-dimensional culture. Elastomer microwells are flipped repeatedly during gel polymerization to ensure an even distribution of buoyant adipocytes.21. Before beginning, calculate the quantities of each reagent needed to prepare a sufficient volume of a 2.5 mg/mL collagen solution for the desired number of elastomer microwells.

Note: While approximately 10 μL of the collagen solution is needed per microwell, prepare an additional 10%–20% to account for volume loss.

22. Place a microcentrifuge tube and all necessary reagents on ice inside a biological safety cabinet. Maintain aseptic technique from this point onward.

23. Add the reagents to the microcentrifuge tube on ice in the following order: 10x media, 1N NaOH, complete media, collagen type I. Mix well between each addition.

Note: Due to its viscosity, use wide bore P200 or P1000 tips to pipette the collagen type I.

24. Ensure the reagents are well mixed and that the collagen solution turns light pink.

CRITICAL: The pH of the collagen type I is variable batch-to-batch. If the solution is orange or yellow after mixing, insufficient 1N NaOH has been added to neutralize the collagen and the solution will not polymerize properly. Add an additional 10% of the initial 1N NaOH volume and mix well. Repeat this process until the solution turns pink. Use pH strips to verify that the pH of the collagen solution is between 7.0 and 7.4 if needed.

25. Keep the resulting collagen solution on ice to prevent premature polymerization.

26. Calculate the total volume of adipocyte-collagen solution needed for the desired number of elastomer microwells. 35 μL of solution is needed per microwell.

Note: Prepare enough adipocyte-collagen solution for two additional microwells to account for volume loss when pipetting.

27. Using a wide bore tip, transfer the required volume of packed adipocytes (75% of the total volume calculated in step 26) to a new microcentrifuge tube.

28. Using a wide bore tip, transfer the required volume of collagen solution (25% of the total volume calculated in step 26) to the same microcentrifuge tube.

29. Using a wide bore tip, gently pipette the packed adipocytes and collagen solution until the resulting mixture looks homogenous.Note: While the collagen solution should be kept on ice to prevent premature polymerization, the packed adipocytes should be kept off ice. For steps 27–28, pipette packed adipocytes into a new microcentrifuge tube and then mix in the cold collagen solution. The larger volume of packed adipocytes will minimize thermal fluctuations and raise the temperature of the collagen solution to begin polymerization.

30. Immediately pipette 35 μL of the adipocyte-collagen solution into the center of each elastomer microwell.

31. Incubate the microwells for 20 min at 21°C to allow the collagen solution to polymerize. Flip the 24-well plate over every 2 min to ensure an even distribution of adipocytes within the gel.

Note: Due to surface tension, the adipocyte-collagen solution will remain within the elastomer microwells when flipped even prior to collagen polymerization.

32. Gently pipette 750 μL of complete media pre-warmed to 37°C down the side of each well, being careful to not dislodge the adipocyte-collagen gel (Figure 2G).

33. Carefully transfer the 24-well plate to a humidified cell culture incubator at 37°C and 5% CO2.

Culture and maintenance of adipocyte-collagen gels

Timing: variable

In this step, adipocyte-collagen gels are cultured ex vivo for up to 5 days. During this time, longitudinal media sampling, treatment with pharmacological inhibitors, and direct or indirect co-culture with other cell types can be performed.34. Transfer the 24-well plate to a biological safety cabinet and replace complete media for the adipocyte-collagen gels every two days or as needed for functional studies.a. Carefully tilt the 24-well plate toward you, allowing the media to collect toward the bottom of each well.

b. Aspirate the media by placing the aspirator tip against the bottom of each well to avoid disturbing the adipocyte-collagen gels.

c. Gently pipette 750 μL of complete media pre-warmed to 37°C down the side of each well, being careful not to dislodge the adipocyte-collagen gels.

35. Return the 24-well plate to the cell culture incubator.

Optional: For direct co-culture studies, other cell types can be resuspended in the calculated volume of complete media when preparing the collagen solution prior to embedding adipocytes. Alternatively, elastomer microwells can be transferred to a 12-well plate using sterile forceps and maintained with additional cells grown in 2D or 3D for indirect co-culture. Use 1.5 mL of complete media when culturing adipocyte-collagen gels in a 12-well format.

Note: Detachment of adipocyte-collagen gels from elastomer microwells can occur during ex vivo culture due to the buoyant force of primary adipocytes. Refer to problem 2 in troubleshooting if detachment is observed.

Assessment of cell viability and contamination in adipocyte-collagen gels

Timing: 1 h

In this step, cell viability and stromal vascular contamination are assessed in a subset of elastomer microwells to ensure successful isolation of primary adipocytes. Isolations yielding gels with low cell viability or significant stromal vascular contamination should be discarded.36. Supplement complete media pre-warmed to 37°C with 2 μg/mL Hoechst 33342 and 2 μg/mL propidium iodide.

37. Transfer the 24-well plate from the cell culture incubator to a biological safety cabinet and move a subset of elastomer microwells for analysis to a new 24-well plate using sterile forceps. Return the remaining microwells to the incubator.

38. Add 750 μL of complete media supplemented with Hoechst 33342 and propidium iodide to each microwell to be analyzed. Place in the cell culture incubator and stain for 30 min protected from light.

39. Aspirate the supplemented media from each well and replace with 750 μL of complete media pre-warmed to 37°C.

40. Move the stained elastomer microwells to an inverted microscope with a humidified chamber at 37°C and 5% CO2 if possible.

41. Using a transfer pipette, place one drop of 1x PBS onto the center of a 22 × 22 mm glass coverslip.

42. Pick up an elastomer microwell by the glass coverslip and invert onto the drop of 1x PBS using forceps. Place the microwell on the drop slowly to avoid trapping air bubbles.

43. Image multiple fields of view for Hoechst 33342, propidium iodide, and bright field for each elastomer microwell using a confocal microscope.

44. Repeat steps 41–43 for each adipocyte-collagen gel.

45. Assess the viability in each sample by dividing the number of live cells (Hoechst-positive, propidium iodide-negative nuclei) by the number of total cells (Hoechst-positive nuclei).

46. Assess the degree of stromal vascular contamination in each sample by looking for clusters of densely packed Hoechst-positive nuclei between adipocytes.

Note: We recommend that the samples analyzed for viability and stromal vascular contamination be discarded and not used for subsequent studies. Refer to problem 3 in troubleshooting if low cell viability is observed. Refer to problem 4 in troubleshooting if significant stromal vascular contamination is observed.

High-resolution imaging of adipocyte-collagen gels

Timing: 2 h

In this step, adipocyte-collagen gels are fixed and stained for subsequent high-resolution imaging on a confocal microscope. While the protocol presented here uses DAPI and Alexa Fluor 488 phalloidin to visualize cell nuclei and F-actin as an instructive example, this protocol can be readily adapted to use other fluorescent dyes and antibodies of interest.47. Using forceps, transfer each adipocyte-collagen gel into a new 24-well plate filled with 1 mL of cold 4% paraformaldehyde (w/v) in 1x PBS per well.

48. Incubate gels for 20 min at 21°C.

49. Aspirate and wash each gel once with 1 mL of 1x PBS.

50. Add 1 mL of 1x PBS supplemented with 1% (w/v) BSA and 0.1% (v/v) Triton-X-100 for 20 min at 21°C.

51. Aspirate and wash each gel once with 1 mL of 1x PBS.

52. Stain each adipocyte-collagen gel for F-actin.a. Prepare 80 μL of staining solution per sample by diluting Alexa Fluor 488 phalloidin to 0.165 μM in 1x PBS supplemented with 1% (w/v) BSA.

b. Aspirate 1x PBS and add 80 μL of staining solution to each elastomer microwell as a drop on top of the adipocyte-collagen gel.

c. Incubate for 1 h at 21°C protected from light.

53. Add 1 mL of 1x PBS to each well and aspirate. Repeat this step one additional time.

54. Stain each adipocyte-collagen gel for double-stranded DNA.a. Prepare 750 μL of staining solution per sample by diluting DAPI to 2.5 μg/mL in 1x PBS supplemented with 1% (w/v) BSA.

b. Aspirate 1x PBS and add 750 μL of staining solution to each well.

c. Incubate for 30 min at 21°C protected from light.

55. Aspirate and wash each gel three times with 1 mL of 1x PBS.

56. Store at 4°C and protect from light until imaged.

Pause point: Fixed and stained samples can be kept at 4°C when protected from light for up to one week prior to imaging.

57. Move stained elastomer microwells to an inverted confocal microscope.

58. Using a transfer pipette, place one drop of 1x PBS onto the center of a 22 × 22 mm glass coverslip.

59. Pick up an elastomer microwell by the glass coverslip and invert onto the drop of 1x PBS using forceps. Place the microwell on the drop slowly to avoid trapping air bubbles.

60. Image multiple fields of view for DAPI and Alexa Fluor 488 phalloidin for each microwell using a confocal microscope.

61. Repeat steps 58–60 for each adipocyte-collagen gel.

Optional: If available, confocal reflectance microscopy can be used to image the collagen fibers surrounding the adipocytes. Note that adipocytes will also generate a reflectance signal due to the different refractive indices of the cytoplasm and lipid droplet.

Expected outcomes

In our experience, resected adipose tissue should yield approximately 30%–40% of the initial tissue volume in primary adipocytes after all steps in this protocol are complete. For example, 2 g of resected adipose tissue (an amount obtainable from pooling the subcutaneous and visceral depots of one adult mouse) would yield 600–800 μL of packed primary adipocytes, sufficient to cast around 20 adipocyte-collagen gels.

The cell viability of adipocyte-collagen gels should remain around 80% or higher for the first 5 days of ex vivo culture (Figures 3A and 3B), after which additional validation will be required. Visible stromal vascular contamination (identified as clusters of densely packed nuclei) should be limited to 1 cluster or fewer per field of view using a 10x objective.Figure 3 Representative images of cell viability in adipocyte-collagen gels

Expected viability of adipocytes isolated from wild type (lean) (A) and ob/ob (obese) (B) mice in collagen gels after 5 days of ex vivo culture. Insets show a representative live and dead adipocyte for each condition. Scale bars = 200 μm.

For high-resolution imaging, visualization of fluorescent dyes or antibodies (Figures 4A–4C) should be possible several hundred micrometers into the adipocyte-collagen gels on a confocal microscope. Beyond this distance, fluorescence signals will dissipate due to light scattering caused by the lipid content of adipocytes.Figure 4 Representative images of adipocyte-collagen gels via high-resolution confocal microscopy

Expected results of adipocyte-collagen gels imaged for cell nuclei (DAPI), F-actin (Alexa Fluor 488 phalloidin), and collagen (confocal reflectance). Images are shown for a single Z-slice (A) and a 180-μm-deep maximum projection (B and C). Note that the confocal reflectance signal has been separated out in the maximum projection to improve visibility. Arrows indicate confocal reflectance signal generated by adipocytes. Dotted lines denote adipocyte boundaries. Scale bar = 100 μm.

Limitations

One major limitation of this protocol stems from the post-mitotic and fragile nature of primary adipocytes. As a result, each experiment requires that primary adipocytes be freshly isolated and immediately used. In addition, while we validated adipocyte viability out to 5 days of ex vivo culture, cell viability will have to be monitored after this time frame and may vary with use case. Collectively, these limitations reduce experimental throughput and prevent longer term studies of primary adipocyte function.

Troubleshooting

Problem 1: Low yield of primary adipocytes

Low yield of primary adipocytes may be caused by several issues. We outline these issues and potential solutions to each below.

Potential solution

• Insufficient amount of initial adipose tissue.○ If an insufficient amount of initial adipose tissue is used, the final yield of primary adipocytes will be low. Use a minimum of 2 g of adipose tissue with this protocol.

• Insufficient mechanical dissociation of adipose tissue.○ If the adipose tissue is not sufficiently minced prior to digestion, the collagenase solution will not have enough surface area to properly digest the tissue. Mince adipose tissue until pieces are smaller in size in step 3.

• Insufficient adipose tissue digestion○ If the adipose tissue is not sufficiently digested, a significant amount of undigested tissue will remain and limit the yield of primary adipocytes. Increase the incubation time or concentration of collagenase type I in step 10 while ensuring these changes do not negatively impact adipocyte viability.

• Insufficient separation of primary adipocytes from undigested tissue.○ If the 200 μm cell strainers become clogged in step 11, primary adipocytes will not pass through into the final cell suspension. Replace the cell strainers as needed and re-strain any adipocytes remaining in the top of clogged strainers.

Problem 2: Detachment of adipocyte-collagen gels from elastomer microwells

Detachment of adipocyte-collagen gels from elastomer microwells during ex vivo culture is caused by the buoyancy of primary adipocytes and may be caused by several issues. We outline these issues and potential solutions to each below.

Potential solution

• Uneven thickness of elastomer microwells.○ If the silicone elastomer solution is not cured on a flat surface, the solid elastomer will have an uneven thickness. Regions of elastomer that are too thin will provide insufficient surface area to promote adhesion of adipocyte-collagen gels following poly(ethyleneimine) functionalization. Ensure elastomer rings are uniform and have a thickness of at least 1 mm in step 14 of before you begin.

• Contamination of elastomer or glass surfaces with dust or debris.○ Significant contamination of elastomer or glass surfaces with dust or debris will interfere with plasma treatment and poly(ethyleneimine) functionalization. Ensure surfaces are clean in step 14 of before you begin. If contamination is visible, wash elastomer rings and coverslips in 70% (v/v) absolute ethanol and then deionized water. Dry completely before use.

• Expired poly(ethyleneimine) or glutaraldehyde solutions.○ Once diluted in a neutral pH buffer, glutaraldehyde will self-crosslink over time and become unable to bond poly(ethyleneimine) to the plasma-treated elastomer and glass surfaces. Prepare a fresh poly(ethyleneimine) working solution every six months and a fresh glutaraldehyde working solution every month in step 19 and step 21 of before you begin.

• Significant contamination of stromal vascular cells in adipocyte-collagen gels.○ While a small amount of stromal vascular contamination is expected, a significant proportion of these cells will shrink the adipocyte-collagen gels over time due to their contractile nature and cause the gels to detach from the elastomer microwells. See problem 4 in troubleshooting to reduce stromal vascular contamination.

• Knocking or shearing of adipocyte-collagen gels.○ Excessive force can dislodge adipocyte-collagen gels from the elastomer microwells. Be gentle when moving well plates containing the gels in all steps and leave a note on the incubator door if needed. When removing or adding media to the wells, maintain a slow flow rate and position the aspirator or pipette tip as far away from adipocyte-collagen gels as possible.

Problem 3: Low cell viability in adipocyte-collagen gels

Low cell viability in adipocyte-collagen gels may be caused by several issues. We outline these issues and potential solutions to each below.

Potential solution

• Prolonged wait times after adipose tissue isolation.○ Primary adipocyte viability decreases over time if left in resected adipose tissue pieces without sufficient diffusion of nutrients or oxygen. Keep resected tissue in tubes on ice and minimize the time between tissue resection and cell isolation. Adipose tissue can be minced into smaller pieces to improve diffusion and then stored on ice if prolonged wait times cannot be avoided.

• Excessive or extreme temperature changes.○ Primary adipocytes are sensitive to temperature change. Place resected adipose tissue in a 50 mL centrifuge tube filled with complete media in step 1. Place the bottom of the tube on ice but do not place tissue directly on ice or at 4°C. If too cold, adipose tissue will congeal, resulting in reduced cell yield and viability. Likewise, avoid unnecessary or repeated temperature fluctuations during tissue processing.

• Excessive adipose tissue digestion.○ Prolonged exposure to collagenase or high concentrations of collagenase in step 10 will negatively impact adipocyte viability. Reduce the collagenase incubation time and concentration as much as possible while maintaining sufficient tissue digestion.

• Excessive handling or centrifugation.○ Excessive handling and centrifugation will lyse adipocytes and reduce cell viability. Always use wide bore pipette tips when handling adipocytes to minimize shear stress and reduce the speed and duration of centrifugation steps as much as possible.

Problem 4: Contamination of adipocyte-collagen gels with a significant proportion of stromal vascular cells

Significant contamination of adipocyte-collagen gels with stromal vascular cells (Figure 5) may be caused by several issues. We outline these issues and potential solutions to each below.Figure 5 Representative images of stromal vascular contamination in adipocyte-collagen gels

Two sample fields of view with stromal vascular contamination. Arrows denote clusters of densely packed nuclei indicative of contamination. Scale bar = 200 μm.

Potential solution

• Insufficient adipose tissue dissociation.○ If adipose tissue pieces are not sufficiently dissociated, stromal vascular cells will not settle out of solution during wash steps and instead contaminate adipocyte-collagen gels. Gradually increase mincing time in step 3 or collagenase incubation time and concentration in step 10 to ensure sufficient tissue dissociation.

• Insufficient straining of undigested tissue.○ If straining the cell suspension after collagenase treatment is unsuccessful, clusters of stromal vascular cells from undigested tissue can contaminate the adipocyte-collagen gels. Ensure that enough cell strainers are used in step 11 and do not attempt to force the cell suspension through a clogged strainer. If contamination persists, filter the cell suspension an additional time through a 200 or 150 μm strainer.

• Insufficient removal of stromal vascular cells from adipocyte suspension.○ During wash steps, gentle mixing causes stromal vascular cells to separate from primary adipocytes due to buoyancy. If significant stromal vascular contamination persists, increase the number of washes with 1x KRH buffer in step 15.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Claudia Fischbach (cf99@cornell.edu).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Garrett F. Beeghly (gfb48@cornell.edu).

Materials availability

This study did not generate or require the use of new unique materials or reagents.

Data and code availability

This study did not generate any datasets or code other than the images provided in the manuscript.

Acknowledgments

We acknowledge support from the 10.13039/100000001 NSF DGE1650441 (G.F.B.), 10.13039/100000054 NCI F31CA278410 (G.F.B.), NCI R01CA259195 (C.F.) and R01CA276392 (C.F.), and the Center on the Physics of Cancer Metabolism NCI 1U54CA210184 (C.F.). We also acknowledge support from the Cornell Biotechnology Resource Center Imaging Facility funded by NYSTEM C029155 , 10.13039/100000002 NIH S10OD018516 , and NIH S10RR025502 . The graphical abstract was created using BioRender.com.

Author contributions

G.F.B. developed the protocol. G.F.B., J.D., and C.F. prepared the manuscript. C.F. supervised the project.

Declaration of interests

The authors declare no competing interests.
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