Human Cells Grown With or Without Substitutes for Fetal Bovine Serum

Abstract

Safety concerns over cell-derived pharmaceutical products being manufactured in supplements of fetal bovine serum (FBS) have ignited pleas to replace FBS. Herein, four newly marketed alternatives to FBS were compared: a xeno-free product called Cell-Ess®, a human platelet lysate marketed as GroPro®, and two mixtures of adult bovine serum varying in their proportions of neonatal growth factors, called Liporo® and FetalGro®. An endothelial cell line (C2BBe1) and a neuronal cell line (SHSY5Y) near confluency in media with 10% FBS were selectively scraped and taken through a 25-day step-wise algorithm to replace FBS, and another human endothelial cell line (HRA-19) was studied to replicate C2BBe1. Cells were stained, counted, and compared for viability, migration, and spheroids. The C2BBe1 and HRA-19 cell lines failed to proliferate in 10% Cell-Ess® but grew in 10% GroPro® or 10% FetalGro® reasonably well compared to reference 10% FBS. With SH-SY5Y, only FetalGro® approached FBS's efficacy. These were all inferior to 11 different branded lots of FBS (positive controls), but five days into switching just amongst the FBS brands, 4 of 11 supported less proliferation than reference FBS in endothelial HRA-19 (p < 0.004). Moreover, neurospheres were enriched in two branded lots of FBS and FetalGro® (each p < 0.004), neurospheres being an unwanted phenotype for any neuronal cell application. Because platelet-derived GroPro® stood out amongst the non-FBS growth supplements to allow proliferation without inducing spheroids, it seems the best (mindful that the cells still grew slower in it compared to FBS). While no perfect replacement was found amongst the alternatives to FBS, the algorithm for switching should be useful in future testing of new alternatives to FBS as the need arises to switch from FBS and expand pharmaceutical products with safety for human use.

Keywords: C2BBe1; SH-SY5Y; fetal bovine serum; neurospheres; xeno-free.

Fig. 1.

Visual appearance of cells in Non-FBS versus FBS media. Cells were observed under phase contrast microscopy (750×) at the end of 25 days (last 5 days) in different media supplements. See Table 1 for an explanation of scrape line and progression of media changes. FBS: fetal bovine serum.

Fig. 2.

Comparison of growth of C2BBe1 cells in non-FBS supplements versus FBS. This was done after the last step in the progression, after the cells had been scraped in swaths and allowed to proliferate for 5 more days in their respective media (Table 1). Cells were stained and counted by cellometry at the end of these 5 days in their media. ‘Total’ refers to the sum of the portions of: attached cells (A) plus floater cells (not shown). Values represent the mean ± SEM. An asterisk indicates the cells in Cell-Ess® yielded statistically lower values than in FBS. None of the other supplements yielded statistical differences from FBS regardless of total cells or attached (A) cells (Seradigm brand). FBS: fetal bovine serum; SEM: standard error of the mean.

Fig. 3.

Comparison of growth of SH-SY5Y cells in non-FBS supplements versus FBS. This was done after the last step in the progression, after the cells had been scraped in swaths and allowed to proliferate for 5 more days in their respective media (Table 1). Cells were stained and counted by cellometry at the end of the 5 days in their media. ‘Total’ refers to the sum of the portions of: attached cells (A) plus floater cells (not shown). Values represent the mean ± SEM. The asterisks indicate that all the alternatives yielded statistically lower values from FBS (Seradigm brand). FBS: fetal bovine serum; SEM: standard error of the mean.

Fig. 4.

Comparison of HRA19a1.aα2F cells grown for 5 days in different brands of sera. Sera was made 10% in basal media and used to culture the HRA19a1.aα2F cell line. Post-scraping cells were stained and counted by cellometry. Values represent the mean ± SEM. The asterisk and lines indicates that ATL BIOL FBS was statistically higher by Tukey’s correction (p < 0.004) than these other sources of serum. ACC: Access Cell Culture; ATCC: American Type Culture Collection; ATL: Atlanta Biologicals; BWST: Biowest; COR: Corning; FBS: fetal bovine serum; FGro: Fetalgro^®; GBM: FBS Benchmark^® from Gemini Bio Products; GFD: FBS Foundation^® from Gemini Bio Products; GIB: FBS from Gibco; LGro: Lipogro^®; RMBI: Rocky Mountain Biologicals; SEM: standard error of the mean; SER: FBS from Seradigm; SIG: Sigma.

Fig. 5.

Comparison of SH-SY5Y cells grown for 5 days in different sources of sera. Sera was made 10% in basal media and used to culture the SH-SY5Y cell line post-scraping. Cells were stained and counted by cellometry. Values represent the mean ± SEM. The asterisk and lines indicate that ATL BIOL FBS was statistically higher by Tukey’s correction (p < 0.004) than the other sources of serum. ACC: Access Cell Culture; ATCC: American Type Culture Collection; ATL: Atlanta Biologicals; BWST: Biowest; COR: Corning; FBS: fetal bovine serum; FGro: Fetalgro^®; GBM: FBS Benchmark^® from Gemini Bio Products; GFD: FBS Foundation^® from Gemini Bio Products; GIB: FBS from Gibco; LGro: Lipogro^®; RMBI: Rocky Mountain Biologicals; SEM: standard error of the mean; SER: FBS from Seradigm; SIG: Sigma.

Fig. 6.

Neurospheres in SH-SY5Y cells grown in different FBS sources. Sera was made 10% in basal media and used to culture the SH-SY5Y cell line 5 days post-scraping the swaths. The wells are the same as in Fig. 5 except counted for neurospheres before they were dispersed for cellometry. Microscopic observations were made of each well at low (100×) magnification in a systematic manner so that all the neurospheres in each well could be counted. Values represent the mean ± SEM. The asterisk indicates ATL BIOL FBS was statistically different in neurosphere numbers by Tukey’s correction (p < 0.004) from the other sources of serum ACC: Access Cell Culture; ATCC: American Type Culture Collection; ATL: Atlanta Biologicals; BWST: Biowest; COR: Corning; FBS: fetal bovine serum; FGro: Fetalgro^®; GBM: FBS Benchmark^® from Gemini Bio Products; GFD: FBS Foundation^® from Gemini Bio Products; GIB: FBS from Gibco; LGro: Lipogro^®; RMBI: Rocky Mountain Biologicals; SEM: standard error of the mean; SER: FBS from Seradigm; SIG: Sigma.