Control of microcarrier feed time by quantitative determination of bead-to-bead transfer during hMSC cultures

Mesenchymal stem cells extracted from the Wharton’s jelly of human umbilical cords (hWJ-MSC) show
increasing interest for cell therapies due to their reduced immunogenicity, high expansion capabilities, fast growth
kinetics and various growth factors synthesis capabilities. hWJ-MSC are adherent-dependent cells meaning their
expansion requires colonization of available adherence surfaces. However, when confluence is almost reached, it
was previously shown that MSC begin to form aggregates that can detach from the microcarriers leading to a
negative impact on cell growth and functionality [1]. To solve this problem an already assessed strategy consists
in the addition of fresh microcarriers during the expansion phase. Until now, no quantitative study describing this
phenomenon have been reported in literature. Besides, addition of fresh new microcarriers should allow to
maintain growing cell, allowing to reach a higher cell density than in a system without microcarriers feed. The
aim of this study is to better control fresh microcarriers addition by quantitatively characterize bead-to-bead
transfer process during MSC cultures. Aiming at this, MSC were cultivated on Cytodex-1 microcarrier in HPLsupplemented
culture medium, in mixed Erlenmeyer flasks. To quantify bead-to-bead transfer homemade
fluorescent Cytodex-1 were used as available adherence surfaces. DAPI nuclei and Live/Dead cell staining were
performed to determine total viable cell number and number of cells per microcarrier. Cell counting was obtained
from computerized analysis of fluorescent microscopy images. Using these methods, microcarrier addition time
was established to maintain constant cell number per microcarrier, avoiding to reach the confluence state. Our
results showed both qualitatively and quantitatively bead-to-bead transfer during microcarrier cultures. By
controlling the addition of fresh microcarriers cell aggregation has been completely prevented. This procedure
allowed an increase of the maximal total cell number by a factor 1.5 in comparison with a culture with only
medium feed addition.
[1]. Ferrari, C., et al., (2012). Limiting cell aggregation during mesenchymal stem cell expansion on
microcarriers. Biotechnology progress, 28(3), 780-787