Umbilical cord Wharton's jelly repeated culture system: a new device and method for obtaining abundant mesenchymal stem cells for bone tissue engineering

Abstract

To date, various types of cells for seeding regenerative scaffolds have been used for bone tissue engineering. Among seed cells, the mesenchymal stem cells derived from human umbilical cord Wharton's jelly (hUCMSCs) represent a promising candidate and hold potential for bone tissue engineering due to the the lack of ethical controversies, accessibility, sourced by non-invasive procedures for donors, a reduced risk of contamination, osteogenic differentiation capacities, and higher immunomodulatory capacity. However, the current culture methods are somewhat complicated and inefficient and often fail to make the best use of the umbilical cord (UC) tissues. Moreover, these culture processes cannot be performed on a large scale and under strict quality control. As a result, only a small quantity of cells can be harvested using the current culture methods. To solve these problems, we designed and evaluated an UC Wharton's jelly repeated culture device. Using this device, hUCMSCs were obtained from the repeated cultures and their quantities and biological characteristics were compared. We found that using our culture device, which retained all tissue blocks on the bottom of the dish, the total number of obtained cells increased 15-20 times, and the time required for the primary passage was reduced. Moreover, cells harvested from the repeated cultures exhibited no significant difference in their immunophenotype, potential for multilineage differentiation, or proliferative, osteoinductive capacities, and final osteogenesis. The application of the repeated culture frame (RCF) not only made full use of the Wharton's jelly but also simplified and specified the culture process, and thus, the culture efficiency was significantly improved. In summary, abundant hUCMSCs of dependable quality can be acquired using the RCF.

Figure 1. The repeated culture frame and structure of umbilical cord.

The repeated culture frame is shown in figures A and B, and figures C and D display in cultures. HE stained cross-section of an umbilical cord (E) shows the veins (F), arteries (G), amnion layer (H) and Wharton’s jelly (I), respectively. The right panel shows the IF staining of CD73 (J), CD90 (K), and CD105 (L) in the Wharton’s jelly. Figures M and N show uniform cell distribution in the suspended tissue block and cell distribution owards the bottom in adherent culture for 7 days, respectively. Scale bars: 2mm in E; 200 um in F–I; 20 um in J–L; and 200 um in M and N.

Figure 2. Immunophenotypic profiles of the T1-P3, T3-P3, and T5-P3 cells.

Figure 3. Multilineage differentiation capacities of the T1-P3, T3-P3 and T5-P3 hUCMSCs were confirmed by histochemical staining (A) and RT-PCR (B).

(A) The formations of mineralization nodes, lipid-rich vesicles, and a cartilaginous extracellular matrix (sulfated proteoglycans) were revealed by Alizarin red, Oil Red O, and Alcian blue staining, respectively. (B) Additionally, cells were harvested for RT-PCR. LPL, lipoprotein lipase; hUCMSCs, human umbilical cord-derived mesenchymal stromal cells. Scale bars: 25 µm in A.

Figure 4. Growth curves of the T1-P3, T3-P3 and T5-P3 hUCMSCs.

The growth curves demonstrate the number of days that the cells were in the lag, log and stationary phases of growth.

Figure 5. Expression of osteogenic differentiation markers.

After a 14-day incubation in osteogenic medium, the expression of osteogenic differentiation markers by T1-P3, T3-P3 and T5-P3 cells was assayed using real-time RT-PCR (A–C). The y-axes (A–C) represent the expression rate of the target genes relative to that of 18S. At the same time, cells were lysed for western blotting. The expression of the osteogenic differentiation markers was detected in the three batches of cells (E). Bars represent the mean ± SD, n = 3. *p>0.05.

Figure 6. Osteogenic evaluation in vivo of TEB containing T5-P3 hUCMSCs (A and C) and T1-P3 hUCMSCs (B and D).

X-ray (E), micro-CT (F), and staining of Masson (A and B) and HE (C and D)were applied to demonstrat new bone formation and neoformative blood vessels in hibateral implanted TEB. Scale bars: 500 um in A–D.