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US-12624322-B2 - Methods for producing mature adipocytes and methods of use thereof

US12624322B2US 12624322 B2US12624322 B2US 12624322B2US-12624322-B2

Abstract

The present invention provides methods and systems which accommodate 3-dimensional adipocyte expansion to produce, e.g., mature adipocytes and synthetic adipose tissue with cellular properties of mature adult organisms, including cell size and cytoarchitecture, and the use of such methods and systems for, e.g., in vitro drug screening and/or toxicity assays, disease modeling, and therapeutic applications.

Inventors

  • Benjamin D. Pope
  • Kevin Kit Parker

Assignees

  • PRESIDENT AND FELLOWS OF HARVARD COLLEGE

Dates

Publication Date
20260512
Application Date
20230303

Claims (20)

  1. 1 . A method for producing mature adipocytes in vitro, comprising: seeding a plurality of progenitor cells into a seeding site; culturing the progenitor cells to produce adipocytes having a first diameter; disposing a fiber layer over the adipocytes; and culturing the adipocytes with the fiber layer until at least one of the adipocytes grows to a second diameter of between about 30 μm and about 300 μm, thereby generating at least one mature adipocyte in vitro.
  2. 2 . The method of claim 1 , wherein the first diameter of the adipocytes is less than 30 μm.
  3. 3 . The method of claim 1 , wherein the second diameter of the adipocytes is between about 50 μm and about 280 μm.
  4. 4 . The method of claim 1 , wherein the fiber layer disposed over the adipocytes comprises a plurality of polymer fibers.
  5. 5 . The method of claim 4 , wherein each of the plurality of polymer fibers have a diameter in a range of about 100 nm to about 1 μm.
  6. 6 . The method of claim 4 , wherein each of the polymer fibers comprises greater than about 50 wt % polycaprolactone (PCL).
  7. 7 . The method of claim 6 , wherein each of the polymer fibers further comprises about 5 to about 49 wt % gelatin.
  8. 8 . The method of claim 4 , wherein each of the polymer fibers includes at least one of proteins, polysaccharides, lipids, or nucleic acids.
  9. 9 . The method of claim 1 , wherein the seeding site includes a pattern of islands, each island having a length, width, or diameter in a range of about 10 μm to about 100 μm, and a center to center spacing between each island being in a range of about 20 μm to about 300 μm, each island comprising one or more layers of an extracellular matrix protein.
  10. 10 . The method of claim 1 , wherein the progenitor cells are pluripotent stem cells, mesenchymal progenitor cells, or pre-adipocytes.
  11. 11 . The method of claim 10 , further comprising: differentiating at least a portion of the mesenchymal progenitor cells into at least one of osteoblasts, chondrocytes, fibroblast, preadipocytes, pericytes, or myocytes.
  12. 12 . A method for controlling hypertrophy of adipocyte cells in culture, the method comprising: disposing a pattern of islands on a support; seeding the pattern of islands with progenitor cells; culturing the progenitor cells to produce adipocytes; and disposing a fiber layer over the adipocytes; the pattern of islands being arranged with a center to center spacing between each island such that when the adipocytes are cultured with the fiber layer disposed over the adipocytes, at least one of the adipocytes grows to a diameter up to the center to center spacing between each island.
  13. 13 . The method of claim 12 , wherein each island from the pattern of islands is comprised of one or more layers of an extracellular matrix protein.
  14. 14 . The method of claim 12 , wherein each island from the pattern of islands is comprised of one or more layers of fibronectin.
  15. 15 . The method of claim 12 , wherein each island of the pattern of islands has a length, width, or diameter in a range of about 10 μm to 100 μm, and the center to center spacing between each island is in a range of about 20 μm to about 300 μm.
  16. 16 . The method of claim 12 , wherein the fiber layer disposed over the adipocytes comprises a plurality of polymer fibers, each of the plurality of polymer fibers having a diameter in a range of about 100 nm to about 1 μm.
  17. 17 . The method of claim 16 , wherein each of the polymer fibers comprises greater than about 50 wt % polycaprolactone (PCL).
  18. 18 . The method of claim 17 , wherein each of the polymer fibers further comprises about 5 to about 49 wt % gelatin.
  19. 19 . The method of claim 16 , wherein each of the polymer fibers includes at least one of proteins, polysaccharides, lipids, or nucleic acids.
  20. 20 . A method for producing a tissue-engineered meat product, the method comprising: seeding a plurality of progenitor cells into a seeding site; culturing the progenitor cells to produce adipocytes; disposing a fiber layer over the adipocytes; culturing the adipocytes with the fiber layer until at least one of the adipocytes grows into a mature adipocyte; and combining the at least one mature adipocyte with an engineered muscle tissue product to produce an engineered meat product.

Description

RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 16/757,133, filed Apr. 17, 2020, which is a U.S. national stage filing, under 35 U.S.C. § 371(c), of International Application No. PCT/US2018/056649, filed on Oct. 19, 2018, which claims the benefit of priority to U.S. Provisional Application No. 62/574,801, filed on Oct. 20, 2017. The entire contents of each of the foregoing applications are incorporated herein by reference. GOVERNMENT SUPPORT The invention was made with government support under HL107440 and DK104165 awarded by National Institutes of Health (NIH). The government has certain rights in this invention. BACKGROUND OF THE INVENTION Adipose tissue is present in vertebrate organisms, performing vital functions including energy storage and metabolism, hormone secretion, mechanical shock absorption, and insulation. Large (up to 290-μm diameter) spherical adipocytes make up the tissue and are typically arranged in an imperfect hexagonal packing architecture resembling honeycomb. The cytoarchitecture, cell size, and lipid composition of adipose tissue contribute to the taste and texture of meat and the shape, stiffness, and structural integrity of body parts. As such, adipose tissue is an important element of tissue engineered food and fat grafting for reconstructive surgery or cosmetic enhancement. Moreover, excess (or inadequate) adipose tissue is often pathogenic and methods to prevent obesity or eliminate its pathogenic consequences are of great interest and value. In vivo, adipocyte hypertrophy is a primary mode of weight gain in which an adipocyte expands up to several thousand-fold in size. Conventional adipocyte culture is performed on generic gelatin-coated 2-dimensional (2-D) polystyrene tissue culture dishes with a limited lifespan (−1 month) and is made up of randomly organized cells with variable differentiation status (˜40% adipocytes) and immature cell size (2 orders of magnitude below mature tissue). However, as soon as lipid accumulation occurs in vitro and the cells are approximately 20 microns in diameter, cell buoyancy increases causing adipocytes to detach from conventional planar culture surfaces, fold-up and lyse well before reaching cell sizes observed in adult humans (Table 1, below; reproduced from Pope et al. (2016) Trends in Cell Biology doi: 10.1016/j.tcb.2016.05.005). Therefore, the current methods for culturing adipocytes do not permit the adipocytes to achieve or maintain in vivo structure or function. At most, adipocytes cultured on gelatin-coated culture dishes achieve a size about 8 times less than mature cells in vivo, e.g., adult human adipocytes, before floating off of the culture surface. Therefore, these cultured adipocytes do not function as they would in an in vivo environment. TABLE 1Adipocyte Sizes in Developing and Adult Humans versus in vitro CultureSubcutaneous and Visceral FatSubcutaneous FatVisceral Fat2DCell SizeFetusaNeonateInfantbLeanObeseLeanObeseCultureDiameter40-5050-8090-13050-13090-27045-11090-20020-60range (μm)Mean cell48 000144 000697 000382 0003 054 000244 0001 596 00065 000volume (μm3)a25-30 weeks gestation.b1-3 months postpartum. Although several platforms have been developed that support adipogenesis, none of these platforms overcome the limitations that 2-dimensional cultures impose on adipocyte size and enable the production of mature adipocytes. Accordingly, there is a need in the art for methods and systems that support adipocyte differentiation and growth to enable the production of mature human adipocytes in vitro. SUMMARY OF THE INVENTION The present invention is based, at least in part, on the discovery of methods and systems that support adipocyte differentiation and growth of mesenchymal progenitor cells and enable adipocyte hypertrophy (an increase in the size of the cells) so that mature human adipocytes are produced in vitro. In particular, methods and systems which accommodate 3-dimensional (3-D) adipocyte expansion and which include protein micropatterning to position and coax differentiation of pre-adipocytes with a nanofibrous network that covers and restrains the micropatterned adipocytes during maturation and hypertrophic growth have been discovered. The network extends the lifespan of adipocyte cultures from one to over six months as well as increased average cell size over 50-fold relative to conventional culture (e.g., 2-D gelatin-coated plate culture). In addition, it has been discovered that the spacing between adipocytes in the micropattern controls the rate of hypertrophy independent of time in culture and, thus, this parameter can be used to tune adipocyte size in the islands. For example, islands spaced 50 um apart are nearly confluent at seeding and produce cell sizes similar to 2D culture, while islands that are spaced 200 um apart can be cultured to 200 um diameter. Accordingly, in one aspect, the present invention provides a method for producing mature adipocytes in