A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

Summary

White adipose tissue (WAT) has critical deficiencies in its current primary culture models, hindering pharmacological development and metabolic studies. Here, we present a protocol to produce an adipose microphysiological system by sandwiching WAT between sheets of stromal cells. This construct provides a stable and adaptable platform for primary WAT culture.

Abstract

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health. Still, there are significant limitations to available primary culture models, all of which have failed to faithfully recapitulate the adipose microenvironment or extend WAT viability beyond two weeks. The lack of a reliable primary culture model severely impedes research in WAT metabolism and drug development. To this end we have utilized NIH's standards of a microphysiologic system to develop a novel platform for WAT primary culture called 'SWAT' (sandwiched white adipose tissue). We overcome the natural buoyancy of adipocytes by sandwiching minced WAT clusters between sheets of adipose-derived stromal cells. In this construct, WAT samples are viable over eight weeks in culture. SWAT maintains the intact ECM, cell-to-cell contacts, and physical pressures of in vivo WAT conditions; additionally, SWAT maintains a robust transcriptional profile, sensitivity to exogenous chemical signaling, and whole tissue function. SWAT represents a simple, reproducible, and effective method of primary adipose culture. Potentially, it is a broadly applicable platform for research in WAT physiology, pathophysiology, metabolism, and pharmaceutical development.

Introduction

Adipose tissue is the primary organ of obesity, which carries direct annual medical costs between $147 billion and $210 billion in the U.S.¹. The accumulation of adipose tissue also contributes to other leading causes of death such as heart disease, type II diabetes, and certain types of cancer². In vitro culture models are essential for metabolic studies and drug development, but current research models of adipose tissue have major deficiencies. Adipocytes are fragile, buoyant, and terminally differentiated cells that will not adhere to cell culture plastics, and therefore cannot be cultured using conventional ....

Protocol

All tasks were performed in adherence to protocols #8759 and #9189, as approved by the IRB Office of LSUHSC-NO. All animal work was performed in adherence to protocol #3285 approved by the IACUC Office at LSUHSC-NO.

1. Seeding of Sandwiching Cell Sheets

NOTE: See Figure 1.

1. Seed ADSCs at approximately 80% confluency in tissue culture plates (6 cm or 6-well plates). For each well of SWAT desired, seed 1 conventional tissue culture well and 1 well of corresponding size on poly(N-isopropylacrylamide (pNIPAAm)-coated tissue culture plastic plate.
   NOTE: Accordingly, a 6-....

Results

Viability of SWAT was initially assessed by serial brightfield imaging of individual WAT clusters (n = 12) over approximately 7.6 weeks. Clusters remained secured in place on the monolayer throughout this time. Slight morphological changes were observed with individual adipocytes warping slightly or shifting positions. However, adipocytes neither become multilocular over time, indicating a lack of dedifferentiation, nor did they exhibit any visible signs of cell death such as cellular ble.......

Discussion

This protocol details the use of ADSCs to sandwich human white adipose tissue; human ADSC cell lines can be isolated via well-established protocols¹⁵. However, the system can be adapted for individualized research requirements (such as using 3T3L-1 cells to sandwich mouse WAT). This process involves handling primary human tissue. Standard safety precautions should be employed; handle human tissues as BSL-2 pathogens (e.g., HIV, HepC). Only handle tissue directly under a BSC. Wear all appr.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10x HBSS	Thermofisher	14185052
Gelatin	Sigma-aldrich	G9391
Collagenase	Sigma-aldrich	C5138
Adenosine	Sigma-aldrich	A9251
DMEM	Thermofisher	11995065
M199 Media	Thermofisher	11043023	Phenol red-free
250 µm Mesh Filter	Pierce	87791
0.2 µm Syringe Filter	Celltreat	229747
5 mL Luer-Lok syringes	BD	309646
Metal Washers			These are simple metal washers and can be bought at any hardware store. They simply add leight weight to the backs of the plugers to ensure even contact between cells and gelatin, while being easy to stock and sterilize. Approximate mass: 6.3 g
Name	Company	Catalog Number	Comments
Heated Equipment
Incubated Orbital Shaker	VWR	10020-988	Samples should be pitched at 45° angle to facilitate collagenase digestion
Heat Block			Set to 37-40 °C and placed under Biosafety Cabinet
Water Bath			Set to ~75 °C
Name	Company	Catalog Number	Comments
Specialized Plastics
Upcell Dishes 6cm of 6-multiwell	Nunc	174902  or 174901	These are commerically available pNIPAAm-coated dishes which can be used to grow the upper sheet of ADSCs. Alternatively, pNIPAAm-coated plates can be produced in-lab. diameter: <6 cm for 6 cm dish, <3.5 cm for 6-well plate; approximate mass: 6.7g for 6 cm dish, 5.1 g for 6-well plate
Plastic Plunger Apparatus			These can be fashioned to fit within desired pNIPAAm-coated plastics (multiwell plates, petri dishes). They are comprised of a simple stem attached to a circular disk. They can be produced in-lab or by any facility that can fashion acrylic plastics