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

Abstract

Background:

Human tissue derived natural extracellular matrix (ECM) has great potential in tissue engineering.

Objective:

We sought to isolate extracellular matrix derived from human umbilical cord and test its potential in tissue engineering.

Methods:

An enzymatic method was applied to isolate and solubilized complete human umbilical cord derived matrix (hUCM). The obtained solution was analyzed for growth factors, collagen and residual DNA contents, then used to coat 2D and 3D surfaces for cell culture application.

Results:

The hUCM was successfully isolated with trypsin digestion to acquire a solution containing various growth factors and collagen but no residual DNA. This hUCM solution can form a coating on 2D and 3D substrates suitable cell culture.

Conclusion:

We developed a new matrix derived from human source that can be further used in tissue engineering.

Keywords

Human matrix tissue engineering growth factors residual DNA stem cells

1. Introduction

One of the focus to generate new biomaterials in regenerative medicine is the development of multifunctional extracellular matrix (ECM) that can mimic a natural environment for cells [1]. To do so, both synthetic and natural polymers have been widely investigated [2]. Natural polymers draw much attention because they usually have less concern about biocompatibility. Collagen and fibronectin are well known and applied in various tissue engineering applications to support cell culture. As the most abundant protein in mammals, collagen can be derived from animal or human tissue. Currently, commercial collagen is usually from rat-tail or human placenta [3,4]. With the concern of xenogeneic rejection, animal derived ECM has no clinical potential and this has led to develop human derived product. Human placenta can generate matrix containing numerous growth factors that have function in angiogenesis [5]. Platelet lysate is another promising source that can be used as clinical-grade supplement for cell expansion [6]. These achievements encourage the sighting of new human tissue sources for tissue engineering.

This study aims to explore human umbilical cord derived matrix (hUCM), which was known to contain huge amount of cytokines [7]. We used an enzymatic method to isolate and solubilized hUCM, characterized the obtained product and assessed its potential in tissue engineering.

2. Methods

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. Results

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)).

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.

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.

3.2. Residual DNA detection

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 $45 \pm 12 nm$ . As assessed by AFM, hUCM can also coat 3D de-endothelialized umbilical artery (Fig. 3(a) and (b)).

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)).

4. Discussion

The discovery of new human derived source for fabricating biomaterials remains a goal to reach in tissue engineering [10]. Besides placenta which is considered as an important and useful source for human derived extracellular matrix for tissue engineering, other sources of healthy human tissues are currently explored. This study reports an enzymatic method to isolate a complex matrix from human umbilical cord. This matrix derived from umbilical cord contained abundant growth factors, and extracellular protein such as collagen. These components clearly provide a suitable environment for stem cells and mature cells adherence and growth. Umbilical cord is considered as a niche for mesenchymal stem cells [11]. When used as a matrix source, the isolation procedure will destroy cells in the tissue and lead to the release of cell DNA. The residual human DNA may induce a host immune response. Only biomaterials which do not contain residual DNA may have a clinical transfer potential [12]. In our study, the procedure was ended by adding FBS to inhibit trypsin reaction. Interestingly, FBS was reported to contain heat-stable nucleases that can eliminate residual DNA [13]. This advantage of FBS allows us to obtain a hUCM without any residual DNA, which indicate the hUCM may be safely used in human regenerative medicine. However, we cannot exclude the risk of xenogeneic reaction due to presence of FBS, even if the percentage used was low. An alternative of FBS will be required to supply a full clinical grade hUCM.

5. Conclusion

Our results revealed an enzymatic method to obtain a complex matrix from human umbilical cord. The hUCM contained various growth factors and different types of collagen, which can provide a biomimetic environment for cell culture. No residual DNA was detected from the hUCM, which makes it a safe product for clinical transfer. The hUCM can easily form a coating on 2D and 3D substrates to support cell culture. This point makes hUCM a strong tool for tissue engineering.

Footnotes

Acknowledgements

We would like to thank the Research Federation 3209 BMCT (Nancy, France). Dan Pan is a PhD student supported by China scholarship council.

Conflict of interest

The authors have no conflict of interest to report.

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