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

Abstract

There is growing interest in human adipose tissue-derived collagen as a replacement for animal origin or synthetic materials. Large amounts of adipose tissues around the kidney are being discarded after kidney surgery; thus, we planned to use this tissue as a potentially ideal source of human collagen. Optimization of the collagen extraction process can contribute to the quality, quantity, supply, and cost of collagen production. To extract highly purified and concentrated collagen from human perirenal adipose tissue, we developed a novel extraction process that is superior to the conventional methods in terms of extraction yield, in vitro cytocompatibility, and physicochemical aspects. The sequence of the process and optimized conditions are as follows: (1) destaining with 0.5% H₂O₂ for 1 h at 4°C, (2) noncollagenous proteins elimination with 1.5 M NaOH for 24 h at 4°C, (3) atelocollagen preparation with 1.0% pepsin for 48 h at 25°C, and (4) collagen hydrolysis with 1.0 M NaOH for 10 min at 60°C. The final product showed significantly increased hydroxyproline (355.26 ± 18.71 pg/mL) and glycine (22.752 μg/mL) content than the conventional acetic acid hydrolyzed collagen (164.13 ± 1.11 pg/mL and 0.947 μg/mL, respectively). The lyophilized collagen showed more specific peaks for amides A, B, I, II, and III on FT-IR analysis and showed a further native architecture of collagen fibrils in scanning electron microscope images. Therefore, the optimized process can be an effective protocol for extracting collagen from human perirenal adipose tissue.

Figure 1

Perirenal adipose tissue-derived extracellular matrix (a) and a concept of collagen extraction process for high purity and good quantity (b).

Figure 2

Pigment destaining efficiency as per the H₂O₂ concentration: (a) morphological observation, (b) total protein and hydroxyproline concentration, and (c) histological comparison (arrow, pigments). Data are presented as mean ± standard deviation values. The values with different superscript letters in a column are significantly different (p < 0.05). Scale bars, 50 μm.

Figure 3

Elimination efficiency of noncollagenous proteins as per the NaOH concentration: (a) morphological observation, (b) total protein and hydroxyproline concentration, and (c) histological comparison. Data are presented as mean ± standard deviation values. The values with different superscript letters in a column are significantly different (p < 0.05). Scale bars, 50 μm.

Figure 4

Atelocollagen preparation efficiency as per pepsin concentration: (a) morphological observation, (b) total protein and hydroxyproline concentration, and (c) histological comparison. Data are presented as mean ± standard deviation values. The values with different superscript letters in a column are significantly different (p < 0.05). Scale bars, 50 μm.

Figure 5

Collagen hydrolysis efficiency as per hydrolyzing agent: (a) morphological observation, (b) total protein and hydroxyproline concentration, (c) cell proliferation comparison with MTT assay, and (d) cell morphological comparison. Data are presented as the mean ± standard deviation values. The values with different superscript letters in a column are significantly different (p < 0.05). Scale bars, 50 μm. Ctrl, uncoated; 0.5 M acetic acid; coated with collagen hydrated with 0.5 M acetic acid; 0.5 M lactic acid, coated with collagen hydrated with 0.5 M lactic acid; 0.5 M NaOH, coated with collagen hydrated with 0.5 M NaOH; 1.0 N HCl, coated with collagen hydrated with 1.0 N HCl.

Figure 6

Comparison of the processed collagen and the crude ECM: (a) hydrolyzed collagen comparison, (b) total protein and hydroxyproline concentration, (c) lyophilized collagen comparison, (d) histological comparison (arrow, pigments), (e) FTIR spectroscopy, and (f) SEM images with the network structure of collagen fibers (magnification, ×400; scale bar, 100 μm). Crude, unprocessed ECM; processed, purified collagen through optimized process. Data are presented as the mean ± standard deviation values. The values with different superscript letters in a column are significantly different (p < 0.05). Scale bars, 50 μm.