Abstract
Adipose tissue is involved in many physiological processes. Therefore, the need for adipose tissue-like analogues either for soft tissue reconstruction or as in vitro testing platforms is undeniable. In this work, we explored the natural features of gellan gum (GG) to recreate injectable stable adipose-like microtissues. GG hydrogel particles with different percentages of polymer (0.5%, 0.75%, 1.25%) were developed and the effect of obtained mechanical properties over the ability of hASCs to differentiate towards the adipogenic lineage was evaluated based on the expression of the early (PPARγ) and late (FABP4) adipogenic markers, and on lipids formation and accumulation. Constructs were cultured in adipogenic induction medium up to 21 days or for six days in induction plus nine days in maintenance media. Overall, no significant differences were observed in terms of hASCs adipogenic differentiation within the range of Young’s moduli between 2.7 and 12.9 kPa. The long-term (up to six weeks) stability of the developed constructs supported its application in soft tissue reconstruction. Moreover, their ability to function as adipose-like microtissue models for drug screening was demonstrated by confirming its sensitivity to TNFα and ROCK inhibitor, respectively involved in the repression and induction of the adipogenic differentiation.
Highlights
Adipose tissue is no longer considered solely an energy storage tissue
Samples were collected with the informed consent of the patients and under a collaboration protocol with 3B’s Research Group, approved by the ethical committees of both institutions. Human adipose derived stem cells (hASCs) were expanded in Alpha Minimum Essential Medium (α-MEM) supplemented with 10% Foetal Bovine Serum (FBS) (Life Technologies, Bleiswijk, The Netherlands) and 1% Antibiotic/Antimycotic (ATB) (Life Technologies, Paisley, UK) in a humidified atmosphere with
After culturing cell-laden hydrogel particles in α-MEM medium for three days, medium was changed to an adipogenic induction medium (IM) consisting of α-MEM supplemented with 10% FBS, 1% ATB, 34 μM of D-pantothenate (Sigma-Aldrich, Sintra, Portugal) and 66 μM of biotin (Sigma-Aldrich, Sintra, Portugal), 200 nM of insulin (Sigma-Aldrich, Sintra, Portugal), 1 μM of dexamethasone (Sigma-Aldrich, Sintra, Portugal), 250 μM of 3-isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich, Sintra, Portugal), and 5 μM of troglitazone (Sigma-Aldrich, Sintra, Portugal)
Summary
Adipose tissue is no longer considered solely an energy storage tissue. It is a complex tissue involved in several biological processes including the endocrine/paracrine regulation of energy metabolism and thermoregulation, that provides key structural protection and support to major organs [1,2]. The combination of adult stem cells from various sources within 3D polymeric structures has been one of the most approached to generate 3D adipose-like tissues The majority of these works have been using 3T3-L1 [4,5,6,7], a murine pre-adipocyte cell line that has limited representation of human tissue physiology. Adult stem cells have been differentiated into the adipogenic lineage in a range of natural/synthetic materials [8,9,10,11] Considerations, such as the mimicking of the dimensions of the fat lobules at the microscale [8] and of adipose tissue mechanical properties, have been addressed from the material’s perspective at the initial stage of development. The obtained adipose-like microtissues can be used for adipose tissue reconstruction of defects from oncologic resection, trauma and congenital abnormalities, or as 3D in vitro tissue analogues for drug screening
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