Human umbilical cord derived matrix: A scaffold suitable for tissue engineering application

2.1. Human umbilical cord derived matrix preparation

Human umbilical cords were collected at the delivery suite at regional maternity hospital of Nancy at the University of Lorraine in accordance with TCG/11/R/011 within 12 h of birth. The whole umbilical cord was cut into small pieces and the connective tissue was dissociated. Umbilical cord connective tissue (2–3 g, wet weight) was digested in 10 mL of trypsin (0.025%) at 37°C for 24 h with magnetic stirring. The stirred suspensions were centrifuged at 16 , 000 g for 10 min. The collected supernatants were filtered (100 μm) and 5 mL of fetal bovine serum (FBS) was added to inhibit trypsin. PBS was added to have a 20 mL final solution. The obtained hUCM solution was conserved at 4°C for further use. The precise concentration of hUCM was calculated as follows: 10 mL of the prepared hUCM solution were lyophilized; 10 mL of solution containing 5 mL of trypsin, 2.5 mL of FBS and 2.5 mL of PBS were also lyophilized. The concentration of hUCM (mg/mL) was calculated by the dry powder weight of total hUCM minus the weight of lyophilized trypsin–FBS–PBS solution.

2.2. Characterization of the hUCM solution

The hUCM solution generated by trypsin digestion was characterized for its cytokine contents by an angiogenesis array kit (R&D systems, France) according to the manufacture’s protocol. Detected spots were analyzed with Image J software for semi-quantitative measurement. Western-blot was performed to detect collagen I in the solution. Rat-tail collagen I was used as a positive control and the trypsin–FBS–PBS mixture as negative control.

In order to check whether the hUCM solution contains residual DNA after trypsin treatment, agarose gel electrophoresis (10%) was performed. The trypsin–FBS–PBS mixture was used as a control. We also assessed the influence of FBS on DNA stability by preparing hUCM solution without adding FBS.

2.3. Elaboration and characterization of the hUCM coating for cell culture

hUCM solution was used to form a coating for cell culture. For 2D coating, the solution was deposited on glass coverslips that were previously treated with SDS–HCl. The glass coverslips were incubated at 37°C for 1 h before being washed twice with PBS. For 3D coating, hUCM solution was injected into de-endothelialized umbilical artery and performed similarly to 2D coating. Atomic force microscopy was employed to evaluate the coating.

2D coating was used to assess its potential for cell culture. Human mesenchymal stem cells and human umbilical vein endothelial cells were isolated according to El Omar et al. [8] and cultured on hUCM coating for 5 days before being marked with phalloidine and DAPI for fluorescent microscopy observation as previously described [9].

3.1. Characterization of human umbilical cord matrix

After 24 hours of magnetic stirring in presence of trypsin, hUCM was digested into a viscous solution (Fig. 1). The concentration of hUCM was then precisely calculated using a lyophilized solution. The influence of trypsin, FBS and PBS was also taken into account during calculation. A cytokine array focused on soluble factors revealed that the solution contained abundant growth factors and ECM proteins, including vascular endothelial growth factors (VEGF), fibroblast growth factors-2 (FGF-2), platelet derived growth factor-AA (PDGF-AA) and transforming growth factor-beta 1 and collagen XVIII (Fig. 2(a)). Collagen I was also detected by western blot in three independently prepared hUCM. We also observed that the neutralization of trypsin by FBS is mandatory, since collagen I was destroyed in absence of trypsin neutralization (Fig. 2(b)).

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Fig. 1.

Human umbilical cord matrix was obtained by trypsin digestion under magnetic stirring, followed by adding FBS. The solution was then lyophilized for precise concentration calculation or further biological characterization.

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Fig. 2.

Characterization of hUCM solution. (a) Biochemical analysis using human angiogenesis array demonstrated growth factors and collagen content. (b) Western-blot using collagen I antibody indicated that hUCM contained collagen I. (c) Residual DNA was determined by agarose gel electrophoresis. No residual DNA was detected in any of the three independently prepared hUCM.

The presence of residual human DNA was next determined. DNA was detected in samples without FBS, but residual DNA was destroyed when FBS was added. Thus, in all the three independently prepared full hUCM, no residual DNA was detected (Fig. 2(c)).

3.3. Cell culture coating derived from hUCM

On 2D glass coverslips, hUCM can form a coating which had a uniform distribution with thickness of 45 ± 12 nm . As assessed by AFM, hUCM can also coat 3D de-endothelialized umbilical artery (Fig. 3(a) and (b)).

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Fig. 3.

A coating can be formed by hUCM on 2D substrate (a) and 3D substrate. The 2D coating can support cell adhesion of human mesenchymal stem cells (c) and human umbilical vein endothelial cells (d). (Colors are visible in the online version of the article.)

Human mesenchymal stem cells and endothelial cells seeded on coated glass slides were showed to adhere as observed on fluorescent images indicating that the new coating is able to support cell adhesion and proliferation (Fig. 3(c) and (d)).