An Alternative to Fetal Bovine Serum for Supplementing Cell Culture Media

Abstract

Fetalgro is a supplemented bovine growth serum formulated as an alternative to fetal bovine serum (FBS) for promoting cell growth. In this study, the suitability of Fetalgro as an FBS alternative is determined for mammalian cell lines. Three lots of Fetalgro were tested alongside two reference lots of FBS on multiple cell lines including Vero, CHO-K1, L-929, and MDCK. Cell growth was characterized through growth curves and multiple passage assays to assess relative performance and growth promoting characteristics of Fetalgro. Results indicate that Fetalgro is a suitable, and in some cases a superior, FBS substitute for most mammalian cell lines, especially when cells are appropriately transitioned to the new media. Included is a discussion of recommendations for the use of Fetalgro with various adherent mammalian cell lines.

Introduction

Cell culture applications are broadly utilized in the life sciences and critically important to advancements in areas such as vaccine development, cancer research, drug discovery, and biopharmaceutical manufacturing. Mammalian cell culture provides an excellent model to understand cell physiology, biochemistry, and drug interactions for biotechnological developments and manufacturing. In order to support proliferation, attachment, and maintenance, most cells in culture depend on serum supplementation. Serum is rich in nutrients such as proteins, growth factors, hormones, amino acids, vitamins, sugars, lipids, trace elements, and other components that support the growth of cells in culture. The most commonly used serum for in vitro research and production applications is fetal bovine serum (FBS). It is essentially unparalleled in its ability to support growth of mammalian cells in culture compared to other serums due to its high levels of nutrients and growth factors, and low levels of antibodies. However, due to an unstable supply and high cost of FBS, there is a push for the development of suitable high quality alternatives. FBS is obtained through the beef processing industry as a by-product. Supply is dictated by beef industry market supply and demand, not fetal bovine serum demand. As a result of this mismatch, pricing for FBS remains volatile and sourcing sometimes difficult. Efforts to overcome the cost and availability challenges involved with using FBS include the use of alternative serum supplemented with growth factors or chemically defined media in which cells are adapted to serum free culture conditions. In this study, we explore the use of Fetalgro, a supplemented bovine serum, as an alternative to FBS. Fetalgro is a Rocky Mountain Biologicals, Inc. proprietary bovine growth serum supplemented with additives and formulated to mimic Fetal Bovine Serum.

Materials and Methods

Cell Lines and Culture Conditions

In order to characterize cell response and ability to maintain growth both long and short term in Fetalgro as compared to FBS, several cell lines were tested using multi-passage cell growth and growth curve assays.

The following cell lines were used to test Fetalgro on growth curves and across multiple passages: Vero (ATCC CCL -81), CHO-K1 (ATCC CCL-61), NCTC clone 929 [L cell, L-929, derivative of Strain L] (ATCC CCL-1), and MDCK.1 (ATCC CRL-2935).

CHO-K1 cells were grown in the base media Gibco F-12K Nutrient Mixture (Catalog number 21127022), and supplemented with 10% FBS. Growth media was treated with penicillin (100 IU/mL) - streptomycin (100 µg/mL) solution (Gibco 15140-122).

All other cell lines were grown in the base media Gibco MEM with Earl’s salts, L-Glutamine (Catalog number 11095080), and supplemented with 10% FBS (RMBIO 20131021FS). Growth media was treated with penicillin (100 IU/mL) and streptomycin (100 µg/mL).

Growth Curve

Cells were recovered from cryopreservation and cultured in growth media containing 10% FBS (RMBIO 20131021FS). Test flasks were seeded after expanding cells over 1-2 passages. Cells were seeded in batches from mid-log growth phase cultures at 2 x 106 cells/flask (2.67 x 104 cells/cm2) in nine T75 flasks for 3 lots of Fetalgro (Lots used include 20161025FG, 20161027FG, 20161031FG, 20150930FG, and 20150302FG) and a reference lot of FBS (Lots used include 20130723FS, 20131124FS). Flasks were counted twice daily over 5 days using the trypan blue exclusion method with a hemocytometer. Data was plotted as cell number per cm2 versus time.

Multi-Passage Cell Growth

Cells were seeded in batches from mid-log growth phase cultures at 2.67 x 104 cells/cm2 with biological duplicates in T75 flasks for 3 lots of Fetalgro (Lots used include 20161025FG, 20161027FG, 20161031FG, 20150930FG, and 20140302FG) and two reference lots of FBS (Lots used include 20130723FS, 20150430FS, 23L161, and Seradigm 007B15). Cells were counted every 3 to 5 days by the trypan blue exclusion method and subcultured to new duplicate flasks at 2.67 x 104 cells/cm2 for 4 to 6 passages. Data was plotted as cell number per cm2 versus passage number.

Results

Vero

Vero cells on Passage 1 show initial lag in growth for cells in Fetalgro compared to FBS (reference lot 20130723FS), with counts at 56% (Lot #20140930FG), 48% (Lot #20161027FG), and 53% (Lot #20161031FG) of the reference lot (Figure 1). The second reference FBS (Lot #23L161) matched the first reference FBS (Lot #20130723FS) well at a 103% growth. Passage 2 showed little change in the lag of cell growth for Fetalgro compared to FBS. However, on passage 3 the difference in growth between FBS and Fetalgro narrowed at 94% (Lot #20140930FG), 70% (Lot #20161027FG), and 81% (Lot #20161031FG) of the reference FBS. By the final count (passage 4), cells in Fetalgro grew comparably to the FBS reference, with an average of 5% higher growth across Fetalgro lots compared to FBS (84% Lot# 20140930FG, 122% Lot# 20161027FG, and 108% Lot# 20161031FG). The Vero growth curve (Figure 2) shows no significant difference between the growth of cells in Fetalgro and FBS.

Chart of Vero Multi-Passage with FBS alternative growth serum.
Figure 1. Vero Multi-Passage
Chart of Vero Growth Curve with FBS alternative growth serum.
Figure 2. Vero Growth Curve

CHO-K1

CHO-K1 cells on Passage 1 show no initial lag in growth for cells in Fetalgro compared to FBS (lot 20130723FS), with counts at 100% (Lot #20161025FG), 91% (Lot #20161027FG), and 135% (Lot #20161031FG) of the reference lot (Figure 3). The second reference FBS (Lot #23L161) matched the first reference FBS (Lot 20130723FS) across multiple passages ranging from 87% to 114% growth. Growth for cells in Fetalgro increased dramatically compared to FBS for concurrent passages (passage 3, passage 4). At passage 4 the average Fetalgro growth was 207% of the reference FBS. The CHO-K1 growth curve (Figure 4) shows a small FBS advantage in growth of cells at 52 hours, with the average Fetalgro at 68% of the FBS. However, by 102 hours Fetalgro outperformed FBS at an average of 158% growth compared to FBS. The growth curve indicates that passing at 4 days rather than 3 days gives higher growth performance for CHO-K1 cells in Fetalgro.

Chart of CHO-K1 Multi-Passage with FBS alternative growth serum.
Figure 3. CHO-K1 Multi-Passage
Chart of CHO-K1 Growth Curve with FBS alternative growth serum.
Figure 4. Figure 4. CHO-K1 Growth Curve

L-929

L-929 cells on Passage 1 (performed at 3 days post-seeding) show overall mild initial lag in growth for cells in Fetalgro compared to FBS (reference lot 20130723FS), with counts at 53% (Lot #20161025FG), 79% (Lot #20140930FG), and 67% (Lot #20150302FG) of the reference lot (Figure 5). The second reference FBS (Seradigm Lot #007B15) matched the first reference FBS (Lot 20130723FS) well at 99% growth. Passage 2 showed a narrowing between differences in growth for Fetalgro compared to FBS, with the average at 84% growth. Later passages show consistency in growth with average Fetalgro at 94% for passage 3, 97% for passage 4, and 112% for passage 5. The L-929 growth curve (Figure 6) shows no significant difference or trends between the growth of cells in Fetalgro and FBS.

Chart of L-929 Multi-Passage with FBS alternative growth serum.
Figure 5. L-929 Multi-Passage
Chart of L-929 Growth Curve with FBS alternative growth serum.
Figure 6. L-929 Growth Curve

MDCK

The MDCK cell line on Passage 1 (5 days post seeding) shows initial lag in growth for cells in Fetalgro, with counts at 66% (Lot #20161025FG), 75% (Lot #20150302FG), and 85% (Lot #20140930FG) of the reference lot 20130723FS (Figure 8). The second reference lot of FBS (Lot#20150430FS) matched the first reference lot well at 97% growth. By passage 2 at 4 days, the cells in Fetalgro show significantly higher growth than those in both FBS reference lots (Fetalgro averaged 218% growth). Subsequent passages show Fetalgro lots maintaining relatively comparable or better growth rates than the FBs reference lots. Passage 3 Fetalgro averaged 113% growth of FBS, and passage 4 averaged 144% growth of FBS. By the 5th passage (counted at 5 days), cells in Fetalgro continued to match or exceed growth compared to the reference FBS with counts at 100% (Lot #20140930FG), 127% (Lot #20150302FG), and 105% (Lot #20161025FG) of the reference lot 20130723FS. The MDCK growth curve (Figure 9) shows that 2 lots of Fetalgro supplemented media allowed MDCK cells to reach peak growth at the end of the exponential growth phase after 69 hours of incubation. At this time point, cells in Fetalgro media were counted at 1.79 x 105 cells/cm2 (Lot #20150302FG), and 2.04 x 105 cells/cm2 (Lot #20140930FG). The third lot of Fetalgro media continued to show an increase in growth after 87 hours post-seeding with a count of 2.02 x 105 cells/cm2 (Lot #20161025FG). At 69 hours, FBS media had cell counts at 1.23 x 105 cells/cm2, but continued to grow exponentially until the 87 hour count. Peak growth before plateau phase for cells in FBS media began after 87 hours of incubation at 1.98 x 105 cells/cm2. By 69 hours post seeding, MDCK cells in Fetalgro have significantly higher growth than the reference FBS by 139%-166% (Figure 8). Fetalgro appears to promote MDCK cell growth better than the reference FBS in the log phase up to optimal time of passage at day 3 (70-72 hours), and then levels off to match FBS in the plateau phase. For FBS, optimal passage at the top of the log growth phase appears to be later, around day 4 where it begins to match growth of cells in the plateau phase for cells in Fetalgro.

Chart of MDCK Multi-Passage with FBS alternative growth serum.
Figure 7. MDCK Multi-Passage
Chart of MDCK Growth Curve with FBS alternative growth serum.
Figure 8. MDCK Growth Curve

Conclusion

Fetalgro is a suitable alternative to FBS for all 4 cell lines tested, with appropriate adaptation protocol for some lines. Most cell lines showed lag in growth compared to FBS until the second or third passage, after which growth matched or exceeded that of cells in FBS. This indicates that after adaptation, the cells in Fetalgro attain growth rates comparable to FBS. L-929 cells grow very consistently and are comparable across passages in Fetalgro compared to FBS.

Vero, L-929, and MDCK cells may be grown in Fetalgro with gradual adaptation over 1 to 3 passages. Adaptation protocol may follow ATCC or cell manufacturer recommendations. Initially, cells may be seeded at high density (a 1:2 split ratio from a confluent culture in mid to late log growth phase) in media containing 10% serum by volume. Half of the serum should be original serum and the other half Fetalgro. Subsequent passages should reduce original serum and replace with Fetalgro until cells are growing in media supplemented at 10% with all Fetalgro. Additionally, passing cells at day 3 rather than day 4 appears to optimize cell proliferation in Fetalgro compared to FBS.

Fetalgro supports higher growth rates than FBS for CHO-K1 cells and this cell line requires no adaptation to Fetalgro. For both growth curve and multi-passage assays, CHO-K1 cells in Fetalgro had a higher growth rate than those in FBS. Cells should be passaged after 4 days for optimal results. Further studies might explore the possibility of achieving comparable growth rates with a lower percent volume concentration in base media of Fetalgro versus FBS. Also, determination of Fetalgro performance on the promotion of protein production in modified CHO cells could further illuminate the suitability of Fetalgro as a CHO-line optimized serum.