Bovine Platelet Lysate as an Alternative Growth Supplement for In Vitro Culture of Triple-Negative Breast Cancer Cells

doi:10.21203/rs.3.rs-4720581/v1

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Bovine Platelet Lysate as an Alternative Growth Supplement for In Vitro Culture of Triple-Negative Breast Cancer Cells

https://doi.org/10.21203/rs.3.rs-4720581/v1

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Cell culture media and associated supplements are vital for the proper growth and development of cells for in vitro cell culture. Fetal bovine serum and human platelet lysate are common additives to cell culture media to promote proliferation. Bovine platelet lysate is a novel supplement obtained from bovine plasma that has the potential to become a cheaper, more ethical alternative to fetal bovine serum and human platelet lysate. In this study, we investigated the proliferative effects of DMEM cell culture media supplemented with either fetal bovine serum, human platelet lysate or bovine platelet lysate for triple-negative breast cancer (MDA-MB-231) cells. For each differentially supplemented media, proliferation, cell diameter, and wound healing capabilities were assessed. We show that cells grown in bovine platelet lysate had a lower proliferation rate than cells grown in either fetal bovine serum or human platelet lysate but had larger cell diameters and comparable wound healing rates.

Growth supplements such as fetal bovine serum or human platelet lysate are crucial components used for in vitro cell culture. Derived from the blood of fetal calves, fetal bovine serum (FBS) contains a rich mixture of growth factors, hormones, amino acids, and other nutrients essential for optimal cell growth and proliferation (Chelladurai, et al. 2021). Additionally, FBS improves the pH buffering capacity of cell culture media and reduces physical damage caused by experimental techniques (Yao and Asayama 2017). However, ethical concerns over the collection of FBS raise controversy, as it is obtained from bovine fetuses when pregnant livestock are mistakenly slaughtered (Lee, et al. 2023). Additionally, FBS production often faces unpredictable supply chain issues, inconsistencies in composition, contamination concerns, and increasing prices (Liu, et al. 2023; Chelladurai, et al. 2021). Several attempts have been made to improve in vitro cell culture with alternatives to FBS; however, reliable replacements have yet to be found (Stout, et al. 2022; Kolkmann, et al. 2020). Therefore, finding a suitable alternative to FBS is necessary.

Human platelet lysate (hPL) has been used as a potential alternative to FBS (Guiotto, et al. 2020). hPL is derived from human blood donations, and the platelet-rich plasma is lysed to release an abundance of growth factors, cytokines and nutrients essential for cell proliferation (Shih and Burnouf 2014). hPL is widely used for clinical applications due to its acellular nature, which reduces immunogenicity concerns (Fonseca, et al 2021). hPL is most commonly utilized for the in vitro cell culture of mesenchymal stem cells (MSCs) for regenerative medicine applications, as its components play an essential role in wound healing and tissue repair (Du, et al. 2022). In addition, hPL has been shown to be an effective alternative to FBS in MCF-7 and T47D breast cancer cell lines (Mentari, et al. 2020; Fazzina, et al. 2016). However, hPL retains many of the same issues associated with FBS, including variability in donor characteristics, shortages, and pathogen contamination (Burnouf, et al. 2016).

Bovine platelet lysate (bPL) is a novel cell culture supplement with the potential to serve as an alternative to FBS or hPL. Bovine plasma is obtained from live cattle in a minimally invasive manner before being processed further into bPL, which contains growth factors, proteins, cytokines, and other nutrients essential for cell growth (Plenty™). Therefore, bPL eliminates many of the ethical concerns associated with FBS. Additionally, production of bPL may not encounter the supply chain issues that FBS faces due to the slaughter-free nature of the product. Furthermore, bPL is more cost-effective than both FBS and hPL. To our knowledge, work has yet to be published on using bPL for in vitro cell culture methods.

In this study, we compared cell culture characteristics of triple-negative breast cancer cells cultured in media supplemented with either FBS, hPL, or bPL. We believe bPL will serve as a feasible alternative to FBS and hPL for in vitro cell culture. We show that cells grown in FBS, hPL, or bPL have comparable cell diameters, proliferation rates, and wound healing rates.

Cell Culture Parameters

The MDA-MB-231 (human triple-negative breast cancer) cell line was obtained from the American Type Culture Collection (ATCC). MDA-MB-231 cells were grown separately in DMEM cell culture media plus 10% FBS and 1% antibiotic-antimycotic media, DMEM plus 10% hPL and 1% antibiotic-antimycotic, or DMEM plus 10% bPL and 1% antibiotic-antimycotic. These cells were incubated at 37°C (5% CO₂). Cells were seeded at ~ 1 ⨯ 10⁵ cells per well in 6-well cell culture plates (Corning Inc., Corning, NY) and additional cell culture media solution was added to achieve a 2 mL total volume solution per well. Cells were given 24 hours after seeding to adhere.

Microscopy and Flow Cytometry

Brightfield microscope images were taken using an EVOS™ M5000 digital inverted microscope (Invitrogen, Carlsbad, CA). All scale bars and in vitro wound diameters were measured and created using the EVOS™ M5000 imaging software. An ORFLO® Moxi Go II flow cytometer (ORFLO Technologies, Ketchum, ID) with single-channel cassettes was used to perform cell counts and cell diameters. When taking cell counts using flow cytometry, data was gated based on the measured mean diameter of the cell ± three standard deviations of the mean based on a sample size of n = 10 from experimentation in previous experiments (Smothers, et al. 2023). Finally, all cell counts at all time points were normalized based on an exact initial seeding of 1 ⨯ 10⁵ cells per well for ease of data interpretation and comparison.

Scratch Test

MDA-MB-231 cells were seeded at 1 ⨯ 10⁵ cells per well in 6-well cell culture plates (Corning Inc., Corning, NY) and allowed to grow for 72 hours after seeding. A wound was created in each culture using a 200 µL micropipette tip to scratch the surface of the well. The initial diameter of the scratch was measured in three locations within the field of view using the EVOS™ M5000 digital inverted microscope (Invitrogen, Carlsbad, CA) imaging software, then averaged. Measurements were taken for each well every 12 hours for 72 hours, then averaged. Data is presented as percent change in diameter.

Data Interpretation and Statistical Analysis

For all quantitative data, a standard one-way ANOVA and unpaired t-test were performed to determine statistical significance.

Cell Proliferation

Cell counts (cells/mL) for TNBC cells were performed (n = 5) for each culture condition at 12 h time points over 72 h. At 60 h, the cell concentration for cells grown in FBS became significantly greater than cells grown in bPL (P = 0.0047) and remained greater at 72 h (P = 0.0087). Cells grown in hPL had a significantly greater concentration than cells grown in bPL starting at just 24 h (P = 0.0028), and the cell concentration of the cells grown in hPL remained higher compared to cells grown in bPL throughout 72 h, with the most significant difference in concentration at 60 h (P = 0.0010).

Cell Size

Cell diameter was also measured for TNBC cells in each culture condition (n = 5). Initially, cells grown in bPL had a larger diameter than cells grown in FBS (P = 0.0455), while cells grown in hPL had a larger initial diameter than cells grown in bPL (P = 0.0175). Cells grown in hPL had inconsistent average diameters over the course of 72-hour growth, whereas cells grown in FBS or bPL had a general increase in cell diameter over time (Supplemental File 1). After 72 h, cells grown in bPL had a larger average diameter (22.70 ± 0.11 µm) than cells grown in either FBS (22.50 ± 0.21 µm) or hPL (22.16 ± 0.33 µm) (P = 0.0877 and P = 0.0079, respectively).

Scratch Test

A scratch test was also performed for TNBC cells (n = 5). At 72 hours, the percent change of wound diameter was decreased by 66.74% for bPL, 76.32% for FBS (P = 0.1911) ,and 72.21% for hPL (P = 0.3349). The only time point with a significant difference in percent change of wound diameter was 60 h, with both cells grown in FBS and hPL having slightly larger changes in diameter than cells grown in bPL (P = 0.0231 and P = 0.0143, respectively).

Figure 2

A scratch test for TNBC cells cultured in either FBS, bPL, or hPL. (A) Brightfield image of TNBC cells cultured in either FBS, bPL, or hPL. (B) Percent change in diameter of wound over 72 h at 12 h time points. (P < 0.05 = *, P < 0.01 = **, P < 0.001 = ***)

In this study, we examined the feasibility of bPL as an alternative to FBS or hPL for in vitro cell culture of triple-negative breast cancer cells. We found that MDA-MB-231 cells grown with bPL have comparable wound healing rates to those grown in FBS and hPL. However, cells grown in bPL had significantly lower proliferation rates than cells grown in hPL and FBS, yet cells grown in bPL had significantly higher cell diameters at the majority of time points. Alongside the ethical, scientific, and economic concerns associated with FBS and hPL, these findings show that bPL is an adequate cell culture supplement alternative.

Cells grown in bPL had slightly lower proliferation rates when compared to FBS and hPL; however, they had a larger cell diameter for several time points, particularly at t = 60 h (Fig. 1). The lower proliferation rate of cells grown in bPL may be partly attributed to their increased size, which gives them less room to expand. Compared to cells grown in bPL, cells grown in FBS had a significantly higher proliferation rate beginning at t = 60 h, whereas culturing in hPL yielded a significantly higher proliferation rate beginning at t = 24 h. The accelerated proliferation rate could be due to differences in growth factor concentration. For example, hPL growth factor concentration is significantly higher than FBS (Mohamed, et al. 2020). The presence of these growth factors may be lesser in bPL; however, the characterization of bPL is not currently publicly available.

We encountered some difficulties while taking measurements for the scratch assay in bPL and hPL-cultured cells. The cells seemed to migrate at a higher frequency when compared to the other conditions, making it difficult to identify the edge of the wound accurately. However, we could only observe this phenomenon qualitatively. For further quantification, cell migration assays could be performed to assess the impact of bPL and hPL on cell migration patterns. No significant difference in wound healing was observed for the scratch assay except at t = 60 h, where cells cultured in FBS or hPL had a higher percent change in diameter than cells in bPL.

Exposure to differentially supplemented media could have contributed to decreased proliferation of bPL-supplemented cells. This could be attributed to differences in cell surface markers; MSCs cultured in different media types express different levels of CD90 and CD105, both of which play an important role in cellular proliferation and tissue formation (Sandhu, et al. 2017; Hagman, et al. 2013). While this effect was not observed for cells cultured using hPL, implementing a “serum starvation” period, where cells are cultured in cell culture media with no additional supplements before experimentation, could be used to produce more consistent results.

Due to the limited availability of supplies, investigation utilized only MDA-MB-231 cells as they were readily available. As mentioned previously, characterization data of bPL is unavailable, limiting the extent to which any comparison could be made regarding types and concentrations of growth factors or other supplements in each media type for cell growth. This information would allow for more accurate and quantitative identification of cell growth in differing growth supplements. Furthermore, during post-analysis of cellular images, increased frequency of multinucleated cells could be observed in cells grown in bPL. While multinucleation is not uncommon in MDA-MB-231 (Mirzayans, et al. 2018; Parekh, et al. 2018), especially in high passage number, we feel that further investigation is warranted. More specifically, DAPI staining and flow cytometry can be performed to quantify multinucleation in cells grown in different media types.

Acknowledgments

We thank Plenty^TM for their support and provided materials. We also thank the Research and Education in Disease Diagnosis and Intervention (REDDI) Lab for allowing the use of their equipment and facilities. Thank you to Creative Inquiry undergraduate students for their assistance in cell culture and other related lab work. Dr. William Bridges of the Clemson University Department of Mathematical and Statistical Sciences was consulted regarding all statistical analyses and assistance with experimental design regarding statistical power. This project was funded by the Clemson University College of Engineering, Computing, and Applied Sciences Opportunity Grant and the Clemson University Office of Creative Inquiry and Undergraduate Research.

M.E.B., M.M.S., A.R.S., and C.A.P. contributed to the conception of the manuscript. A.R.S. designed project methodology. M.E.B., M.M.S., C.C.S., and K.L.M. performed the experiments. M.E.B. and M.M.S. mentored and instructed Creative Inquiry undergraduate students. A.R.S. curated and analyzed data. M.E.B. and M.M.S. drafted the manuscript. M.E.B., M.M.S., and A.R.S. contributed to close analysis and editing of the manuscript. All authors contributed to, read, and approved the submitted manuscript.

Disclosures

The authors of this publication received material research support from Plenty^TM which is developing products related to research described in this publication. Specifically, Plenty^TM provided samples of bovine platelet lysate and the authors independently investigated its viability for in vitro cell culture. The terms of this arrangement have been reviewed and approved by Clemson University in accordance with its policy on objectivity in research.

Competing Interests

The authors of this publication received material research support from Plenty which is developing products related to research described in this publication. Specifically, Plenty provided samples of bovine platelet lysate and the authors independently investigated its viability for in vitro cell culture. The terms of this arrangement have been reviewed and approved by Clemson University in accordance with its policy on objectivity in research.

Author Contribution

M.B., M.S., A.S., and C.P. contributed to the conception of the manuscript. A.S. designed project methodology. M.B., M.S., C.S., and K.M. performed the experiments. M.B. and M.S. mentored and instructed Creative Inquiry undergraduate students. A.S. curated and analyzed data. M.B. and M.S. drafted the manuscript. M.B., M.S., and A.S. contributed to close analysis and editing of the manuscript. All authors contributed to, read, and approved the submitted manuscript.

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Competing interest reported. The authors of this publication received material research support from Plenty which is developing products related to research described in this publication. Specifically, Plenty provided samples of bovine platelet lysate and the authors independently investigated its viability for in vitro cell culture. The terms of this arrangement have been reviewed and approved by Clemson University in accordance with its policy on objectivity in research.

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Bovine Platelet Lysate as an Alternative Growth Supplement for In Vitro Culture of Triple-Negative Breast Cancer Cells

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Abstract

Figures

Introduction

Materials and Methods

Cell Culture Parameters

Microscopy and Flow Cytometry

Scratch Test

Data Interpretation and Statistical Analysis

Results

Cell Proliferation

Cell Size

Scratch Test

Discussion

Declarations

References

Additional Declarations

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