MSCs Isolation Efficiency and MSCs Characteristics

Our original cohort of donor samples was n = 110, but 39 of these samples were used to establish a standard protocol for CT collection, transportation, and processing for freezing. We chose a nonenzymatic method in order to reduce the use collagenase therefore reducing the expense, time of preparation of the cord (stripping out the vessels and tying the ends together), and possible problems with clinical approval. With a focus on the nonenzymatic method, we determined that an ideal protocol with less manipulation of the CT would be advantageous and we wanted to determine if we could obtain cells from frozen tissues. Preparing cells from fresh tissue then freezing them for later use has a much higher upfront costs for the tissue banks compared with storing frozen tissue and only isolating cells when required. Although public banks are not currently storing CT or CT cells this should change as clinical trials using CT‐MSCs are increasing. Thus, keeping the cost down will make CT‐MSCs storage feasible for public and family banks. The method of finely chopping fresh CT and plating the cells for outgrowth presented us with two main questions. (a) Can this be successfully applied to frozen tissue? (b) How large can a tissue fragment be to be frozen successfully and still allow for the outgrowth of MSCs? Increasing the size of the tissue fragment would mean less chopping, which means less labor and therefore less cost. There was no precedent for the experiment. We ran experiments on 39 donor samples to establish the optimized protocol prior to the 71 reported in this article.

We expected that with thick pieces of tissue, only a portion of the tissue would freeze properly. We wanted to determine whether enough of the tissue would survive to yield cells in an explant culture. We tested CT sections of 4 mm, 6 mm, and 8 mm thickness. We froze the whole section as described in the article. After 7 days, the tissue was thawed, fixed, embedded, and sectioned for histology. We found that the 8 mm thick sections had necrotic centers but the 4 mm and 6 mm sections did not. We then thawed 4 mm and 6 mm sections in explant cultures. Keeping with the idea of minimal manipulation, we chose to cut each tissue section into just four pieces and plate each one into a separate well of a 6‐well plate.

The data for these initial 39 samples were similar to the data reported here for the 71 samples processed in our final protocol format. Cells were observed migrating from the piece of tissue at various days. Outgrowth was defined as five or more cells. The time to confluence in the well of the 6‐well plate for the first plating only also varied with each sample. However, after the first passage, the proliferation rate of each sample was surprisingly consistent. The piece of tissue would not be removed until confluence was reached. We found that we could move the piece of tissue to a fresh well and more cells would migrate out. We only tested this to three transfers. The finalized protocol consisted of cutting whole cords into 6 mm sections and freezing in cryopreservant for 7 days minimum before thawing, dividing the tissue section into four pieces and plating, as described in the Materials and Methods. These preliminary experiments (n = 39) provided the basis to run another 71 samples that are reported on here.

The next 71 samples were all collected and processed using our newly established Standard Operating Procedure (SOP). The protocol that resulted from this study included transporting the CT in a closed specimen container without liquid and processing the CT without the use of enzymatic digestion within 24 hours of collection. The tissues was then frozen and after a minimum of 7 days in liquid nitrogen, thawed, and the cells were isolated and characterized as described in the Materials and Methods section. All donor samples collected and processed this way resulted in viable and proliferating cells that were further characterized. Cell outgrowth appeared 5–38 days (average 14.5 days) after the initial plating for 100% of donors (n = 71; Table 2). Cells that grew out of the tissue were heterogeneous in size, had a spindle appearance, and had a typical MSC appearance. The cells migrated away from the tissue in a scattered formation. As the cells proliferated and became confluent, they aligned to form a parallel, organized sheet of cells (Fig. 1A). For all samples, the cells were not passaged until the 35 mm well was 80% confluent (∼120,000 cells per cm2; designated as passage 1). At this point, all samples exhibited similar proliferation characteristics, reaching confluence every 4 days after being plated 1:4 to keep an initial plating density of ∼3,000 cells per cm2 from passage 1 on. This rate of cell proliferation remained consistent for all donors followed through to P10 (n = 20).

Table 2. Average time to cell outgrowth. Variation in first cell outgrowth ranges from 5 days to 38 days, with an average of 14.5 days. CT no. Day first passage CT no. Day first passage CT no. Day first passage CT no. Day first passage 1 8 21 22 41 5 61 38 2 14 22 22 42 17 62 17 3 8 23 16 43 38 63 17 4 10 24 15 44 17 64 7 5 14 25 14 45 20 65 7 6 10 26 27 46 5 66 7 7 14 27 16 47 5 67 7 8 10 28 16 48 20 68 7 9 8 29 14 49 8 69 7 10 10 30 16 50 20 70 7 11 35 31 14 51 5 71 7 12 10 32 16 52 5 Average 14.5 13 8 33 16 53 27 14 15 34 17 54 20 15 27 35 14 55 27 16 8 36 16 56 5 17 14 37 5 57 27 18 17 38 38 58 5 19 16 39 5 59 5 20 14 40 5 60 27

Figure 1 Open in figure viewer PowerPoint Immunophenotypic analysis of cord tissue (CT)‐mesenchymal stromal cells (MSCs). MSCs from multiple donors were analyzed at three different passages: passage 2 (n = 40), passage 5 (n = 20), and passage 10 (n = 20) to determine their MSC profile. (A): The cells migrate from the piece of CT and have the typical MSCs shape. As the cells reach confluence, they become aligned and elongated. (B): The percentage of cells expressing CD73, CD44, and CD90 remained consistent throughout the 10 passages, whereas the percentage of cells expressing CD105 declined after passage 5. Statistically significant differences are denoted by * (p < .05).

Immunophenotypic cell surface analysis of MSCs obtained from explant culture was done using flow cytometry at passage 2 (n = 40), passage 5 (n = 20), and passage 10 (n = 20; Supporting Information Fig. S1). All samples analyzed illustrated similar phenotypic cell surface expression profiles. We observed that all samples contained hematopoietic cells at the earliest passage analyzed (P2). The number of these cells decreased with passage indicating that the culture media selected for MSCs growth only (Fig. 1B).

Cell passaging did not affect MSCs marker expression, with the exception of CD105. There was no difference in the profiles of CD44 with 99.7% ± 0.059% positive cells at p2, 99.5% ± 0.374% at p5, and 99.2% ± 0.372% at p10. The same trend was observed for CD90+ cells; p2 = 99.7% ± 0.0787%, p5 = 99.8% ± 0.05029%, and p10 = 99.0% ± 0.568% and for CD73+ cells; p2 = 99.7% ± 0.0579%, p5 = 99.5% ± 0.0146%, and p10 = 99.2% ± 0.3415%. CD105 (Endoglin) expression decreased after passage 5. The intensity of CD105 expression of the positive cells remained high, but the frequency of cells expressing CD105 was reduced from p2 (95.4% ± 0.845%) to p5 (94.3% ± 0.435%) to p10 (84.7% ± 3.525%; p < .05).

A subset of CT‐MSCs was chosen randomly and tested for their ability to differentiate into adipose and osteoblasts. All samples tested (n = 7) differentiated into both cell types with equal efficiency (Fig. 2).