Optimization of extracellular matrix extraction from human perirenal adipose tissue

Abstract

Human adipose tissue includes useful substrates for regenerative medicine such as the extracellular matrix (ECM), but most perirenal fat tissue is wasted after kidney surgery. Since a lot of adipose tissue can be procured after a kidney, we extracted ECM from human perirenal adipose tissue and optimized the extraction process. To verify the efficacy for ECM extraction, we compared the products in several steps. Perirenal adipose tissue was either finely homogenized or underwent crude manual dissection. The amount of extracted ECM was quantified with ELISA for verification of the initial tissue downsizing effect. To validate the drying effect for fast and complete delipidation, tissues were prepared in a dry or wet phase, and residual lipids were visualized with Oil-Red-O staining. The extracted lipid was assayed at each time point to quantify the appropriate delipidation time. To select the optimal decellularization method, tissues were treated with physical, chemical, or enzymatic method, and the residual cell debris were identified with histological staining. The biochemical properties of the ECM extracted by the above methods were analyzed. The ECM extracted by fine homogenization showed a significantly enhanced amount of collagen, laminin and fibronectin compared to the crude dissection method. The dried tissue showed fast and complete lipid elimination compared to the wet tissue. Complete delipidation was achieved at 45 min after acetone treatment. Additionally, 1% triton X-100 chemical treatment showed complete decellularization with well-preserved collagen fibers. Biochemical analysis revealed preserved ECM proteins, a high cell proliferation rate and normal cell morphology without cell debris or lipids. The established process of homogenization, drying, delipidation with acetone, and decellularization with Triton X-100 treatment can be an optimal method for ECM extraction from human perirenal adipose tissue. Using this technique, human perirenal adipose tissue may be a valuable source for tissue engineering and regenerative medicine.

Optimized Collagen Extraction Process to Obtain High Purity and Large Quantity of Collagen from Human Perirenal Adipose Tissue

“…After obtaining approval from the Institutional Research Ethics Review Board of the Kyungpook National University Hospital (IRB# KNUH 2019-08-008-002), human perirenal adipose tissue was obtained from healthy donors who had undergone donor nephrectomy at the Kyungpook National University Hospital, Deagu, Korea. Perirenal adipose tissue-derived ECM was obtained as described in our previous report [ 6 ]. In brief, the perirenal adipose tissue was dissected and dried.…”

“…Gradually, the range of fat sources has expanded, and visceral fat has been utilized. Among the visceral fat, for the first time, we reported the effective extraction method of ECM from the human perirenal adipose tissue [ 6 ]. Perirenal fat tissues are simultaneously resected during the kidney surgeries, and the resected tissue is disposed as medical waste.…”

“…The amount of discarded human perirenal adipose tissue is continuously rising owing to the increasing number of kidney donations and kidney cancer. There are two limitations in the use of perirenal adipose tissue, as follows: (1) the perirenal adipose tissue-derived ECM is a complex material composed of materials, such as collagen, fibronectin, vitronectin, and laminin, although collagen is present in the highest proportion [ 6 ]. (2) The ratio of ECM components may vary, depending on the individual.…”

Decellularized ECM hydrogels: prior use considerations, applications, and opportunities in tissue engineering and biofabrication

“…22 Chloroform, acetone, and methanol have been reported as solvents for lipid extraction. 23,24 Triton X-100, a non-ionic detergent, has also been used to disturb the lipid–lipid and lipid–protein interactions. 9,25 Another example is bone tissue which is often exposed to a demineralization step, generally performed by acid extraction, to remove mineral content from the tissue.…”

“…22 Chloroform, acetone, and methanol have been reported as solvents for lipid extraction. 23,24 Triton X-100, a non-ionic deter-Fig. 1 Preparation and applications of dECM hydrogels.…”