Growth Of Chinese Hamster Ovary Cells Research Articles

Depletion of endogenous miRNA-378-3p increases peak cell density of CHO DP12 cells and is correlated with elevated levels of ubiquitin carboxyl-terminal hydrolase 14

miRNAs are potent molecular regulators of cellular behaviour. The manipulation of these small non-coding RNAs has been used to enhance industrially relevant phenotypes in Chinese Hamster Ovary (CHO) cells. We investigated the stable depletion of six miRNAs; miR-204-5p, 338-3p, 378-3p, 409-3p, 455-3p and 505-3p, robustly associated with cell growth rate from a previous profiling study. Inhibition of endogenous miR-378-3p function by miRNA-sponge-decoy improved peak cell density by 59%. Quantitative label free LC–MS/MS proteomic analysis of the fractionated cell cultures at day 4 and 8 of batch culture found 216 cytosolic and 114 membrane-associated proteins differentially expressed with stable miR-378-3p depletion. qRT-PCR of 8 genes; Clic4, Hnrnpa1, Prdx1, Actn4, Usp14, Srxn1, Canx and Gnb1, with unidirectional differential protein expression over the two time points of analysis was carried out. In-silico predictive algorithms; TargetScan and miRDB, were used to decipher possible direct targets of miR-378-3p. The Ubiquitin carboxyl-terminal hydrolase 14 (Usp14) protein was identified in the cytosolic fractions at both timepoints as differentially expressed with an increased abundance of 1.58-fold in the miR-378-3p depleted cells on day 8. Usp14 is a deubiquitinase (DUB) with previous reports of its up-regulation leading to increased proliferation of cancer cells. Overexpression of Usp14 in CHO cells had significant effects on cell growth supporting a role for Usp14 in the increased peak cell density seen with miR-378-3p depletion. This study highlights miR-378-3p as a novel engineering candidate for improving CHO cell growth. The use of subcellular fractionation also improved proteome coverage in the identification of novel miRNA targets.

Mitochondrial aconitase is a key regulator of energy production for growth and protein expression in Chinese hamster ovary cells.

Mammalian cells like Chinese hamster ovary (CHO) cells are routinely used for production of recombinant therapeutic proteins. Cells require a continuous supply of energy and nutrients to sustain high cell densities whilst expressing high titres of recombinant proteins. Cultured mammalian cells are primarily dependent on glucose and glutamine metabolism for energy production. The TCA cycle is the main source of energy production and its continuous flow is essential for cell survival. Modulated regulation of TCA cycle can affect ATP production and influence CHO cell productivity. To determine the key metabolic reactions of the cycle associated with cell growth in CHO cells, we transiently silenced each gene of the TCA cycle using RNAi. Silencing of at least four TCA cycle genes was detrimental to CHO cell growth. With an exception of mitochondrial aconitase (or Aco2), all other genes were associated with ATP production reactions of the TCA cycle and their resulting substrates can be supplied by other anaplerotic and cataplerotic reactions. This study is the first of its kind to have established key role of aconitase gene in CHO cells. We further investigated the temporal effects of aconitase silencing on energy production, CHO cell metabolism, oxidative stress and recombinant protein production. Transient silencing of mitochondrial aconitase inhibited cell growth, reduced ATP production, increased production of reactive oxygen species and reduced cell specific productivity of a recombinant CHO cell line by at least twofold.

The Expression Pattern of the Phosphoproteome Is Significantly Changed During the Growth Phases of Recombinant CHO Cell Culture.

Post-translational modification of proteins by reversible phosphorylation plays a pivotal role in regulating key cellular processes including transcription, translation, cell proliferation, differentiation, apoptosis, and signal transduction. Despite the importance of the phosphorylation level of regulation, little work has been carried out on the phosphoproteomic characterization of Chinese hamster ovary (CHO) cells in bioprocess-relevant conditions. Growth control strategies are often used to prolong culture duration and increase specific productivity; however, the cellular mechanisms and regulatory pathways underlying growth strategies are poorly understood in CHO cells. Phosphorylation changes are dynamic and will respond to changes in culture conditions; this may reflect the status of the cells with respect to growth and viability of the culture. Herein, this study uses a phosphopeptide enrichment strategy in conjunction with LC-MS/MS to carry out a large-scale differential phosphoproteomic analysis of IgG producing CHO DP12 cells at various phases of growth in serum-free suspension batch culture to characterize dynamic changes to the phosphoproteome with changing culture conditions. In total over the various growth phases, 3777 differentially expressed unique phosphopeptides are identified from 1415 differentially expressed unique phosphoproteins. Analysis of the whole cell lysate without phosphopeptide enrichment over the various growth phases revealed the differential expression of 834 unique proteins, with an overlap of 188 proteins between the proteomic and phosphoproteomic analyses. The inclusion of phosphoproteomic data significantly improves proteome coverage but also gives insights into the post-translational level of regulation during cellular growth of recombinant CHO cells.

High glucose and low specific cell growth but not mild hypothermia improve specific r-protein productivity in chemostat culture of CHO cells.

In the biopharmaceutical sector, Chinese hamster ovary (CHO) cells have become the host of choice to produce recombinant proteins (r-proteins) due to their capacity for correct protein folding, assembly, and posttranslational modification. However, the production of therapeutic r-proteins in CHO cells is expensive and presents insufficient production yields for certain proteins. Effective culture strategies to increase productivity (qp) include a high glucose concentration in the medium and mild hypothermia (28–34 °C), but these changes lead to a reduced specific growth rate. To study the individual and combined impacts of glucose concentration, specific growth rate and mild hypothermia on culture performance and cell metabolism, we analyzed chemostat cultures of recombinant human tissue plasminogen activator (rh-tPA)-producing CHO cell lines fed with three glucose concentrations in feeding media (20, 30 and 40 mM), at two dilution rates (0.01 and 0.018 1/h) and two temperatures (33 and 37 °C). The results indicated significant changes in cell growth, cell cycle distribution, metabolism, and rh-tPA productivity in response to the varying environmental culture conditions. High glucose feed led to constrained cell growth, increased specific rh-tPA productivity and a higher number of cells in the G2/M phase. Low specific growth rate and temperature (33 °C) reduced glucose consumption and lactate production rates. Our findings indicated that a reduced specific growth rate coupled with high feed glucose significantly improves r-protein productivity in CHO cells. We also observed that low temperature significantly reduced qp, but not cell growth when dilution rate was manipulated, regardless of the glucose concentration or dilution rate. In contrast, we determined that feed glucose concentration and consumption rate were the dominant aspects of the growth and productivity in CHO cells by using multivariate analysis.

The Long Non‐Coding RNA Transcriptome Landscape in CHO Cells Under Batch and Fed‐Batch Conditions

The role of non-coding RNAs in determining growth, productivity, and recombinant product quality attributes in Chinese hamster ovary (CHO) cells has received much attention in recent years, exemplified by studies into microRNAs in particular. However, other classes of non-coding RNAs have received less attention. One such class are the non-coding RNAs known collectively as long non-coding RNAs (lncRNAs). The authors have undertaken the first landscape analysis of the lncRNA transcriptome in CHO using a mouse based microarray that also provided for the surveillance of the coding transcriptome. The authors report on those lncRNAs present in a model host CHO cell line under batch and fed-batch conditions on two different days and relate the expression of different lncRNAs to each other. The authors demonstrate that the mouse microarray is suitable for the detection and analysis of thousands of CHO lncRNAs and validated a number of these by qRT-PCR. The authors then further analyzed the data to identify those lncRNAs whose expression changed the most between growth and stationary phases of culture or between batch and fed-batch culture to identify potential lncRNA targets for further functional studies with regard to their role in controlling growth of CHO cells. The authors discuss the implications for the publication of this rich dataset and how this may be used by the community.

13 C Flux Analysis Reveals that Rebalancing Medium Amino Acid Composition can Reduce Ammonia Production while Preserving Central Carbon Metabolism of CHO Cell Cultures.

13 C metabolic flux analysis (MFA) provides a rigorous approach to quantify intracellular metabolism of industrial cell lines. In this study, 13 C MFA was used to characterize the metabolic response of Chinese hamster ovary (CHO) cells to a novel medium variant designed to reduce ammonia production. Ammonia inhibits growth and viability of CHO cell cultures, alters glycosylation of recombinant proteins, and enhances product degradation. Ammonia production was reduced by manipulating the amino acid composition of the culture medium; specifically, glutamine, glutamate, asparagine, aspartate, and serine levels were adjusted. Parallel 13 C flux analysis experiments determined that, while ammonia production decreased by roughly 40%, CHO cell metabolic phenotype, growth, viability, and monoclonal antibody (mAb) titer were not significantly altered by the changes in media composition. This study illustrates how 13 C flux analysis can be applied to assess the metabolic effects of media manipulations on mammalian cell cultures. The analysis revealed that adjusting the amino acid composition of CHO cell culture media can effectively reduce ammonia production while preserving fluxes throughout central carbon metabolism.

Targeting miRNAs with CRISPR/Cas9 to Improve Recombinant Protein Production of CHO Cells.

MicroRNAs with their unique ability to target hundreds of genes have been highlighted as powerful tools to improve bioprocess behavior of cells. The common approaches to stably deplete miRNAs are the use of sponge decoy transcripts or shRNA inhibitors, which requires the introduction and expression of extra genetic material. As an alternative, we implemented the CRISPR/Cas9 system in our laboratory to generate Chinese hamster ovary (CHO) cells which lack the expression of a specific miRNA for the purpose of functional studies. To implement the system, miR-27a/b was chosen as it has been shown to be upregulated during hypothermic conditions and therefore may be involved in controlling CHO cell growth and recombinant protein productivity. In this chapter, we present a protocol for targeting miRNAs in CHO cells using CRISPR/Cas9 and the analysis of the resulting phenotype, using miR-27 as an example. We showed that it is possible to target miRNAs in CHO cells and achieved ≥80% targeting efficiency. Indel analysis and TOPO-TA cloning combined with Sanger sequencing showed a range of different indels. Furthermore, it was possible to identify clones with no detectable expression of mature miR-27b. Depletion of miR-27b led to improved viability in late stages of batch and fed-batch cultures making it a potentially interesting target to improve bioprocess performance of CHO cells.

The effect of hydrogen peroxide produced during ultraviolet disinfection of CHO cell culture media

Abstract Ultraviolet (UV) irradiation is being considered for protection against viral contamination in cell culture media. Hydrogen peroxide (H2O2) produced by UV irradiation has been suggested as the cause for poor cell growth in irradiated media, but this hypothesis has not been carefully evaluated. The impact of H2O2 on Chinese Hamster Ovary (CHO) cell culture medium was compared with the impact of UV irradiation. Media composition was analyzed via nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography mass spectrometry (LCMS), and cell growth in treated media was also evaluated. Although addition of H2O2 to medium caused significant changes in pyruvate, formate, acetate, and sarcosine concentrations, there was less effect on CHO cell growth compared with irradiation. UV irradiation caused other changes in composition that did not occur as a result of H2O2 addition. Catalase inhibited the effects of adding H2O2 to the media, but catalase added before irradiation did not affect most irradiation-induced changes, even though catalase retained activity. In conclusion we found that while H2O2, which can be generated as a result of UV-irradiation, may be the cause of some changes in medium composition, it does not directly account for impaired CHO cell growth after high UV doses.

MiR-143 targets MAPK7 in CHO cells and induces a hyperproductive phenotype to enhance production of difficult-to-express proteins.

In recent years, the number of complex but clinically effective biologicals such as multi-specific antibody formats and fusion proteins has increased dramatically. However, compared to classical monoclonal antibodies (mAbs), these rather artificially designed therapeutic proteins have never undergone millions of years of evolution and thus often turn out to be difficult-to-express using mammalian expression systems such as Chinese hamster ovary (CHO) cells. To provide access to these sophisticated but effective drugs, host cell engineering of CHO production cell lines represents a promising approach to overcome low production yields. MicroRNAs (miRNAs) have recently gained much attention as next-generation cell engineering tools. However, only very little is known about the capability of miRNAs to specifically increase production of difficult-to-express proteins. In a previous study we identified miR-143 amongst others to improve protein production in CHO cells. Thus, the aim of the present study was to examine if miR-143 might be suitable to improve production of low yield protein candidates. Both transient and stable overexpression of miR-143 significantly improved protein production without negatively affecting cell growth and viability of different recombinant CHO cells. In addition, mitogen-activated protein kinase 7 (MAPK7) was identified as a putative target gene of miR-143-3p in CHO cells. Finally, siRNA-mediated knock-down of MAPK7 could be demonstrated to phenocopy pro-productive effects of miR-143. In summary, our data suggest that miR-143 might represent a novel genetic element to enhance production of difficult-to-express proteins in CHO cells which may be partly mediated by down-regulation of MAPK7. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1046-1058, 2017.

The enhancement of antibody concentration and achievement of high cell density CHO cell cultivation by adding nucleoside.

Recently, with the dramatic increase in demand for therapeutic antibodies, Chinese hamster ovary (CHO) cell culture systems have made significant progress in recombinant antibody production. Over the past two decades, recombinant antibody productivity has been improved by more than 100-fold. Medium optimization has been identified as an important key approach for increasing product concentrations. In this study, we evaluated the effects of deoxyuridine addition to fed-batch cultures of antibody-expressing CHO cell lines. Furthermore, we investigated the effects of combined addition of deoxyuridine, thymidine, and deoxycytidine. Our results suggest that addition of these pyrimidine nucleosides can increase CHO cell growth, with no significant change in the specific production rate. As a result of the increased cell growth, the antibody concentration was elevated and we were able to achieve more than 9g/L during 16days of culture. Similar effects of nucleoside addition were observed in fed-batch cultures of a Fab fragment-expressing CHO cell line, and the final Fab fragment concentration was more than 4g/L. This nucleoside addition strategy could be a powerful platform for efficient antibody production.

Impact of Dissolved Oxygen during UV-Irradiation on the Chemical Composition and Function of CHO Cell Culture Media.

Ultraviolet (UV) irradiation is advantageous as a sterilization technique in the biopharmaceutical industry since it is capable of targeting non-enveloped viruses that are typically challenging to destroy, as well as smaller viruses that can be difficult to remove via conventional separation techniques. In this work, we investigated the influence of oxygen in the media during UV irradiation and characterized the effect on chemical composition using NMR and LC-MS, as well as the ability of the irradiated media to support cell culture. Chemically defined Chinese hamster ovary cell growth media was irradiated at high fluences in a continuous-flow UV reactor. UV-irradiation caused the depletion of pyridoxamine, pyridoxine, pyruvate, riboflavin, tryptophan, and tyrosine; and accumulation of acetate, formate, kynurenine, lumichrome, and sarcosine. Pyridoxamine was the only compound to undergo complete degradation within the fluences considered; complete depletion of pyridoxamine was observed at 200 mJ/cm2. Although in both oxygen- and nitrogen-saturated media, the cell culture performance was affected at fluences above 200 mJ/cm2, there was less of an impact on cell culture performance in the nitrogen-saturated media. Based on these results, minimization of oxygen in cell culture media prior to UV treatment is recommended to minimize the negative impact on sensitive media.

Improving expression of recombinant human IGF-1 using IGF-1R knockout CHO cell lines.

Chinese Hamster Ovary (CHO) cells are widely used for the large-scale production of recombinant biopharmaceuticals. However, attempts to express IGF-1 (a mutated human Insulin-like growth factor 1 Ea peptide (hIGF-1Ea mut)) in CHO cells resulted in poor cell growth and low productivity (0.1-0.2 g/L). Human IGF-1 variants negatively impacted CHO cell growth via the IGF-1 receptor (IGF-1R). Therefore knockout (KO) of the IGF-1R gene in two different CHO cell lines as well as knockdown (KD) of IGF-1R in one CHO cell line were performed. These cell line engineering approaches decreased significantly the hIGF-1 mediated cell growth inhibition and increased productivity of both KO CHO cell lines as well as of the KD CHO cell line. A productivity increase of 10-fold at pool level and sevenfold at clone level was achieved, resulting in a titer of 1.3 g/L. This data illustrate that cell line engineering approaches are powerful tools to improve the yields of recombinant proteins which are difficult to produce in CHO cells.

Real-time monitoring of the physiological state of CHO cells in bioreactor by using a dual spectroscopic strategy

Background Bioprocesses of mammalian cell culture have become essential in pharmaceutical fields for the production of recombinant therapeutic proteins such as monoclonal antibodies or for tissue therapy. Following the FDA’s recommendation to apply Process Analytical Technology (PAT) approach as the mean to control production processes and ensure the quality of the end-product for patients, Near Infra-Red (NIR)and dielectric spectroscopic technologies have gained great attention over the past decade as online tools for cellular bioprocesses monitoring [1,2]. In such processes, cells are one of the most critical parameter, because their physiological state directly impacts the final product titer through their productivity as well as the product quality. Animal cells are typically classified into viable or dead entities via Trypan Blue exclusion technique, but lysed cells are not accounted for, although they could represent a significant proportion of the cell population in bioreactors. In addition to reducing productivity, cell lysis causes the release of intracellular proteases and glycosidases, which can degrade secreted recombinant protein of interest. Therefore, they should be considered for optimal control of processes. In this work, we propose the first online strategy combining NIR and dielectric spectroscopies for in-depth characterization of cellular growth and physiology of CHO (Chinese Hamster Ovary) cells cultivated in bioreactors.

6-Substituted Pyrrolo[2,3-d]pyrimidine Thienoyl Regioisomers as Targeted Antifolates for Folate Receptor α and the Proton-Coupled Folate Transporter in Human Tumors.

2-Amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidine antifolate thiophene regioisomers of AGF94 (4) with a thienoyl side chain and three-carbon bridge lengths [AGF150 (5) and AGF154 (7)] were synthesized as potential antitumor agents. These analogues inhibited proliferation of Chinese hamster ovary (CHO) sublines expressing folate receptors (FRs) α or β (IC50s < 1 nM) or the proton-coupled folate transporter (PCFT) (IC50 < 7 nM). Compounds 5 and 7 inhibited KB, IGROV1, and SKOV3 human tumor cells at subnanomolar concentrations, reflecting both FRα and PCFT uptake. AGF152 (6) and AGF163 (8), 2,4-diamino-5-substituted-furo[2,3-d]pyrimidine thiophene regioisomers, also inhibited growth of FR-expressing CHO and KB cells. All four analogues inhibited glycinamide ribonucleotide formyltransferase (GARFTase). Crystal structures of human GARFTase complexed with 5 and 7 were reported. In severe combined immunodeficient mice bearing SKOV3 tumors, 7 was efficacious. The selectivity of these compounds for PCFT and for FRα and β over the ubiquitously expressed reduced folate carrier is a paradigm for selective tumor targeting.

Using simple models to describe the kinetics of growth, glucose consumption, and monoclonal antibody formation in naive and infliximab producer CHO cells.

Despite their practical and commercial relevance, there are few reports on the kinetics of growth and production of Chinese hamster ovary (CHO) cells—the most frequently used host for the industrial production of therapeutic proteins. We characterize the kinetics of cell growth, substrate consumption, and product formation in naive and monoclonal antibody (mAb) producing recombinant CHO cells. Culture experiments were performed in 125 mL shake flasks on commercial culture medium (CD Opti CHO™ Invitrogen, Carlsbad, CA, USA) diluted to different glucose concentrations (1.2–4.8 g/L). The time evolution of cell, glucose, lactic acid concentration and monoclonal antibody concentrations was monitored on a daily basis for mAb-producing cultures and their naive counterparts. The time series were differentiated to calculate the corresponding kinetic rates (rx = d[X]/dt; rs = d[S]/dt; rp = d[mAb]/dt). Results showed that these cell lines could be modeled by Monod-like kinetics if a threshold substrate concentration value of [S]t = 0.58 g/L (for recombinant cells) and [S]t = 0.96 g/L (for naïve cells), below which growth is not observed, was considered. A set of values for μmax, and Ks was determined for naive and recombinant cell cultures cultured at 33 and 37 °C. The yield coefficient (Yx/s) was observed to be a function of substrate concentration, with values in the range of 0.27–1.08 × 107 cell/mL and 0.72–2.79 × 106 cells/mL for naive and recombinant cultures, respectively. The kinetics of mAb production can be described by a Luedeking–Piret model (d[mAb]/dt = αd[X]/dt + β[X]) with values of α = 7.65 × 10−7 µg/cell and β = 7.68 × 10−8 µg/cell/h for cultures conducted in batch-agitated flasks and batch and instrumented bioreactors operated in batch and fed-batch mode.

Inhibitory Effect of Excessive Glucose on Its Biochemical Pathway and the Growth of Chinese Hamster Ovary (CHO) Cells

GRAPHICAL ABSTRACT

Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media.

The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.

Overexpression of microRNAs enhances recombinant protein production in Chinese hamster ovary cells.

MicroRNAs (miRNAs) are short, non-coding RNAs that can negatively regulate expression of multiple genes at post-transcriptional levels. Using miRNAs to target multiple genes and pathways is a promising cell-engineering strategy to increase recombinant protein production in mammalian cells. Here, we identified miRs-17, -19b, -20a, and -92a to be differentially expressed between high- and low- monoclonal antibody-producing Chinese hamster ovary (CHO) cell clones using next-generation sequencing and quantitative real-time PCR. These miRNAs were stably overexpressed individually and in combination in a high-producing clone to assess their effects on CHO cell growth, recombinant protein productivity and product quality. Stably transfected pools demonstrated 24-34% increases in specific productivity (qP) and 21-31% increases in titer relative to the parental clone, without significant alterations in proliferation rates. The highest protein-producing clones isolated from these pools exhibited 130-140% increases in qP and titer compared to the parental clone, without major changes in product aggregation and N-glycosylation profile. From our clonal data, correlations between enhanced qP/titer and increased levels of miRs-17, -19b, and -92a were observed. Our results demonstrate the potential of miRs-17, -19b, and -92a as cell-engineering targets to increase recombinant protein production in mammalian cells.

Poly-γ-glutamic acid enhances the quality of recombant erythropoietin produced by CHO cells

The effect of poly-γ-glutamic acid (γPGA), which is produced by Bacillus sp., on the production of recombinant erythropoietin (rEPO) by Chinese hamster ovary (CHO) cells in suspension culture was evaluated. The growth, viability, and productivity of recombinant CHO cells were investigated in a chemically defined medium with 50 and 500 kD γPGAs at 0.075% or with Pluronic F68. Cell growth with the two γPGAs was lower than that with Pluronic F68 but significantly higher than that without any additive (control). The effect of additives on rEPO productivity was 50 kDa γPGA > 500 kDa γPGA > Pluronic F68 > control. Using EPO-dependent F-36E cells, we found that the effect of additives on rEPO quality was 500 kDa γPGA > 50 kDa γPGA > control > Pluronic F68. γPGA has an enhancement effect on the quality of rEPO produced by CHO cells.

Serum-free medium optimization based on trial design and support vector regression.

The Plackett-Burman design and support vector machine (SVM) were reported to be used on many fields such as some feature selections, protein structure prediction, or forecasting of other situations. Here, with suspension adapted Chinese hamster ovary (CHO) cells as the object of study, a serum-free medium for the culture of CHO cells in suspension was optimized by this method. Support vector machine based on genetic algorithm was used to predict the growth rate of CHO and prove the results from the trial designs. Experimental results indicated that ZnSO4, transferrin, and bovine serum albumin (BSA) were important ones. The same conclusion was arrived at when the support vector regression model analyzed the experimental results. With the methods mentioned, the influence of 7 medium supplements on the growth of CHO cells in suspension was evaluated efficiently.