Characterization of Human AB Serum for Mesenchymal Stromal Cell Expansion

Abstract

Background: So far, using human blood-derived components appears to be the most efficient and safest approach available for mesenchymal stromal cell (MSC) expansion. In this paper, we report on the characterization of human AB serum (AB HS) produced by using different plasma sources, and its use as an alternative supplement to MSC expansion.

Methods: Two plasma sources were used for AB HS production: plasma removed from whole blood after 24 h of collection (PC > 24 h) and plasma, cryoprecipitate reduced (PCryoR). The biochemical profile and quality of the produced AB HS batches were analyzed and their ability to support MSC cell growth after different storage times (0, 3, 6, 9 and 12 months) was evaluated.

Results: The two plasma sources used showed similar characteristics regarding biochemical constituents and quality parameters and were effective in promoting MSC growth. MSCs cultured in medium supplemented with 10% AB HS presented similar doubling times and cumulative population doublings when compared to the 10% fetal bovine serum(FBS)-supplemented culture while maintaining immunophenotype, functional features, and cytogenetic profile.

Conclusion: Overall, the results indicate that AB HS is an efficient FBS substitute and can be used for at least 12 months after production without impairing cell proliferation and quality.

Keywords: Cell expansion; Fetal bovine serum; Human AB serum; Mesenchymal stromal cells.

Fig. 1

MSC morphology cultured in AB HS from PCryoR (A) and PC > 24 h (B) and FBS (C); (D) DT of MSCs grown during passages 5-7 in culture medium supplemented with FBS (n = 6) and AB HS batches (n = 12) with different post-production storage times (0, 3, 6, 9 and 12 months). Results shown as mean ± SD.

Fig. 2

Growth kinetics of MSC using culture medium supplemented with FBS and AB HS batches right after production (A) and after 3 (B), 6 (C), 9 (D) and 12 months (E) of storage at −20 °C (n = 6). Results shown as mean ± SD.

Fig. 3

Evaluation of the effect of AB HS storage time in the MSC growth obtained from (A) PCryoR (n = 6) and (B) PC > 24 (n = 6). Results shown as mean ± SEM.

Fig. 4

Immunophenotypic profile and inhibition of T-lymphocyte proliferation of MSC cultured in the culture medium supplemented with FBS (n = 3) and AB HS obtained from PCryoR (n = 3) and PC > 24 h (n = 3) after 12 months of storage. Results shown as mean ± SD.

Fig. 5

Cell differentiation of MSCs cultivated with FBS, AB HS PC > 24 h, or AB HS PCryoR and stored for different times 0, 3, 6, 9 and 12 months. Differentiation of MSCs to adipocytes, osteocytes, and chondrocytes was induced.

Fig. 6

A representative karyotype of MSC culture on alpha-MEM culture medium supplemented with AB HS (12 months of storage). Analysis of 20 metaphases revealed 46,XX. The karyogram illustrated in the figure presents a level of resolution of 625-band stage.