Plant Cell Suspension Cultures Research Articles

Regulatory effects and mechanisms of hormones on the growth and rosmarinic acid synthesis in the suspension-cultured cells of Origanum vulgare

Plant cell suspension culture has many advantages such as short production cycle and independence of natural conditions, and therefore shows a good application prospect in the production of plant secondary metabolite. In this paper, the regulatory effects of hormones on the growth and RosA (rosmarinic acid) synthesis of the cultured O. vulgare cells were comprehensively investigated, meanwhile, the mechanisms of the regulation were explored using integrated multi-omics analysis. The hormone screening and optimization tests revealed that 6-BA and NAA showed a significant promotion effect on cell growth, but 2,4-D displayed an inhibitory effect on growth, while KT, TDZ, ZT and IBA had no significant effect. The preferred hormone combination for cell growth was 4.0 mg/L NAA and 0.5 mg/L 6-BA, under which cell biomass was up to 20.25 g/L. Nine hormones, including KT, ZT, TDZ, 2,4-D, IBA, GA3, ETH, MeJA, and Spm, inhibited the synthesis of RosA in the cells, while ABA exhibited a significant promotion on the synthesis. The yield of RosA reached 283.54 mg/L under ABA induction, which was 3.47 times higher than that of the control. The integrated transcriptome and metabolome analysis showed that the expression levels of TFs (transcriptional regulators) such as GT-3A, MYB61, and WRKY72A were significantly up-regulated under ABA treatment, which in turn significantly promoted the expression of key genes for RosA synthesis like 4-coumarate CoA-ligase, cytochrome P450 monooxygenase, hydroxyphenylpyruvate reductase, abscisic-aldehyde oxidase and xanthoxin dehydrogenase, ultimately leading to a significant increase in RosA synthesis in cells.

Arabinogalactan-Proteins as Boron-Acting Enzymes, Cross-Linking the Rhamnogalacturonan-II Domains of Pectin

Most pectic rhamnogalacturonan-II (RG-II) domains in plant cell walls are borate-bridged dimers. However, the sub-cellular locations, pH dependence, reversibility and biocatalyst involvement in borate bridging remain uncertain. Experiments discussed here explored these questions, utilising suspension-cultured plant cells. In-vivo pulse radiolabelling showed that most RG-II domains dimerise extremely quickly (<4 min after biosynthesis, thus while still intraprotoplasmic). This tallies with the finding that boron withdrawal causes cell wall weakening within 10–20 min, and supports a previously proposed biological role for boron/RG-II complexes specifically at the wall/membrane interface. We also discuss RG-II monomer ↔ dimer interconversion as monitored in vitro using gel electrophoresis and a novel thin-layer chromatography method to resolve monomers and dimers. Physiologically relevant acidity did not monomerise dimers, thus boron bridge breaking cannot be a wall-loosening mechanism in ‘acid growth’; nevertheless, recently discovered RG-II trimers and tetramers are unstable and may thus underpin reversible wall loosening. Dimerising monomers in vitro by B(OH)3 required the simultaneous presence of RG-II-binding ‘chaperones’: co-ordinately binding metals and/or ionically binding cationic peptides. Natural chaperones of the latter type include highly basic arabinogalactan protein fragments, e.g., KHKRKHKHKRHHH, which catalyse a reaction [2 RG-II + B(OH)3 → RG-II–B–RG-II], suggesting that plants can ‘enzymically’ metabolise boron.

Performance of Malaysian kenaf Hibiscus cannabinus callus biomass and exopolysaccharide production in a novel liquid culture

ABSTRACTThe versatility of a well-known fibrous crop, Hibiscus cannabinus (kenaf) is still relatively new to many. Kenaf’s potential applications, which can be extended even into critical industries such as pharmaceutical and food industries, have always been overshadowed by its traditionally grown fiber. Therefore, this study aimed to venture into the biotechnological approach in reaping the benefits of kenaf through plant cell suspension culture to maximize the production of kenaf callus biomass (KCB) and exopolysaccharide (EPS), which is deemed to be more sustainable. A growth curve was established which indicates that cultivating kenaf callus in suspension culture for 22 days gives the highest KCB (9.09 ± 1.2 g/L) and EPS (1.1 ± 0.02 g/L). Using response surface methodology (RSM), it was found that sucrose concentration, agitation speed, and naphthalene acetic acid (NAA) concentration can affect the production of KCB and EPS significantly (p < 0.05) while 2,4-dichlorophenoxy acetic acid (2,4-D) was deemed insignificant. To maximize the final yield of KCB and EPS, the final optimized variables are 50 g/L sucrose, 147.02 rpm, and 2 mg/L of NAA. To conclude, the optimized parameters for the cell suspension culture of kenaf callus serve as the blueprint for any sustainable large-scale production in the future and provide an alternative cultivating method to kenaf traditional farming.

Impact of Phylogenetically Diverse Bacterial Endophytes of Bergenia pacumbis on Bergenin Production in the Plant Cell Suspension Cultures.

Plant in vitro cultures are potential sources for secondary metabolites. However, low productivity is often a major drawback for industrial application. Elicitation is an important strategy to improve product formation in vitro. In this context, endophytes are of special interest as biotic elicitors due to their possible interaction with the metabolism of the host plant. A total of 128 bacterial endophytes were isolated from the medicinal plant Bergenia pacumbis and taxonomically classified using 16S rRNA gene sequencing. Five strains belonging to different genera were grown in lysogeny broth and tryptic soy broth medium and cells as well as spent media were used as elicitors in cell suspension cultures of B. pacumbis. Production of the main bioactive compound bergenin was enhanced 3-fold (964 µg/g) after treatment with cells of Moraxella sp. or spent tryptic soy broth medium of Micrococcus sp. These results indicate that elicitation of plant cell suspension cultures with endophytic bacteria is a promising strategy for enhancing the production of desired plant metabolites.

Elicitation of flavonoid biosynthesis in cell suspension cultures of Haplophyllum virgatum by sodium nitroprusside

Plant cell suspension culture, along with elicitor application, is a suitable technique to obtain large amounts of bioactive compounds. Haplophyllum virgatum is an Iranian endemic plant species. Sodium nitroprusside (SNP) is widely used as a nitric oxide (NO) donor in studies on plants to investigate the physiological roles of NO. Here, the physiological responses were investigated in cell suspension cultures of H. virgatum in the presence of different concentrations of SNP (0, 100, 150, and 200 µM) at various time points (0, 8, 12, 24, 48, 72, and 168 h). The transcript levels of two flavonoid biosynthetic enzymes, including chalcone synthase (CHS) and chalcone isomerase (CHI), and an important transcription factor (R2R3-MYB), were measured in the SNP-treated cells. The content of rutin (the main flavonoid in the Rutaceae family) was also evaluated. Nitric oxide significantly decreased the hydrogen peroxide and malondialdehyde contents by increasing the proline and enzymatic and non-enzymatic antioxidants in the treated cells. Meanwhile, NO significantly elicited flavonoid production, including rutin. These results highlight the effects of NO on flavonoid biosynthesis in H. virgatum suspension cells, probably through gene expression regulation in and upstream of the flavonoid biosynthetic pathway.

Enhancement of recombinant human keratinocyte growth factor 1 protein production in transgenic hazelnut (Corylus avellana L.) plant cell suspension cultures under RAmy3D inducible promoter

Enhancement of recombinant human keratinocyte growth factor 1 protein production in transgenic hazelnut (Corylus avellana L.) plant cell suspension cultures under RAmy3D inducible promoter

Biopotentials of Collagen Scaffold Impregnated with Plant-Cell-Derived Epidermal Growth Factor in Defective Bone Healing.

The combination of scaffolds with recombinant human epidermal growth factor (rhEGF) protein can enhance defective bone healing via synergistic activation to stimulate cellular growth, differentiation, and survival. We examined the biopotentials of an rhEGF-loaded absorbable collagen scaffold (ACS) using a mouse model of calvarial defects, in which the rhEGF was produced from a plant cell suspension culture system because of several systemic advantages. Here, we showed a successful and large-scale production of plant-cell-derived rhEGF protein (p-rhEGF) by introducing an expression vector that cloned with its cDNA under the control of rice α-amylase 3D promoter into rice calli (Oryza sativa L. cv. Dongjin). Implantation with p-rhEGF (5 μg)-loaded ACSs into critical-sized calvarial defects enhanced new bone formation and the expression of osteoblast-specific markers in the defected regions greater than implantation with ACSs alone did. The potency of p-rhEGF-induced bone healing was comparable with that of Escherichia coli-derived rhEGF protein. The exogenous addition of p-rhEGF increased the proliferation of human periodontal ligament cells and augmented the induction of interleukin 8, bone morphogenetic protein 2, and vascular endothelial growth factor in the cells. Collectively, this study demonstrates the successful and convenient production of p-rhEGF, as well as its potency to enhance ACS-mediated bone regeneration by activating cellular responses that are required for wound healing.

Production of Secondary Metabolites from Cell Cultures of Sageretia thea (Osbeck) M.C. Johnst. Using Balloon-Type Bubble Bioreactors.

Sageretia thea is used in the preparation of herbal medicine in China and Korea; this plant is rich in various bioactive compounds, including phenolics and flavonoids. The objective of the current study was to enhance the production of phenolic compounds in plant cell suspension cultures of Sageretia thea. Optimum callus was induced from cotyledon explants on MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D; 0.5 mg L-1), naphthalene acetic acid (NAA, 0.5 mg L-1), kinetin (KN; 0.1 mg L-1) and sucrose (30 g L-1). Browning of callus was successfully avoided by using 200 mg L-1 ascorbic acid in the callus cultures. The elicitor effect of methyl jasmonate (MeJA), salicylic acid (SA), and sodium nitroprusside (SNP) was studied in cell suspension cultures, and the addition of 200 µM MeJA was found suitable for elicitation of phenolic accumulation in the cultured cells. Phenolic and flavonoid content and antioxidant activity were determined using 2,2 Diphenyl 1 picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethybenzothiazoline-6-sulphonic acid (ABTS), ferric reducing antioxidant power (FRAP) assays and results showed that cell cultures possessed highest phenolic and flavonoid content as well as highest DPPH, ABTS, and FRAP activities. Cell suspension cultures were established using 5 L capacity balloon-type bubble bioreactors using 2 L of MS medium 30 g L-1 sucrose and 0.5 mg L-1 2,4-D, 0.5 mg L-1 NAA, and 0.1 mg L-1 KN. The optimum yield of 230.81 g of fresh biomass and 16.48 g of dry biomass was evident after four weeks of cultures. High-pressure liquid chromatography (HPLC) analysis showed the cell biomass produced in bioreactors possessed higher concentrations of catechin hydrate, chlorogenic acid, naringenin, and other phenolic compounds.

Plant Cell Cultures: Biofactories for the Production of Bioactive Compounds

Plants have long been exploited as a sustainable source of food, flavors, agrochemicals, colors, therapeutic proteins, bioactive compounds, and stem cell production. However, plant habitats are being briskly lost due to scores of environmental factors and human disturbances. This necessitates finding a viable alternative technology for the continuous production of compounds that are utilized in food and healthcare. The high-value natural products and bioactive compounds are often challenging to synthesize chemically since they accumulate in meager quantities. The isolation and purification of bioactive compounds from plants is time-consuming, labor-intensive, and involves cumbersome extraction procedures. This demands alternative options, and the plant cell culture system offers easy downstream procedures. Retention of the metabolic cues of natural plants, scale-up facility, use as stem cells in the cosmetics industry, and metabolic engineering (especially the rebuilding of the pathways in microbes) are some of the advantages for the synthesis and accumulation of the targeted metabolites and creation of high yielding cell factories. In this article, we discuss plant cell suspension cultures for the in vitro manipulation and production of plant bioactive compounds. Further, we discuss the new advances in the application of plant cells in the cosmetics and food industry and bioprinting.

Enhancing the Production of Hydrolytic Enzymes in Elicited Tamarillo (Solanum betaceum Cav.) Cell Suspension Cultures.

Plant cell suspension cultures are widely used as a tool for analyzing cellular and molecular processes, metabolite synthesis, and differentiation, bypassing the structural complexity of plants. Within the range of approaches used to increase the production of metabolites by plant cells, one of the most recurrent is applying elicitors capable of stimulating metabolic pathways related to defense mechanisms. Previous proteomics analysis of tamarillo cell lines and cell suspension cultures have been used to further characterize and optimize the growth and stress-related metabolite production under in vitro controlled conditions. The main objective of this work was to develop a novel plant-based bioreactor system to produce hydrolytic enzymes using an elicitation approach. Based on effective protocols for tamarillo micropropagation and plant cell suspension culture establishment from induced callus lines, cell growth has been optimized, and enzymatic activity profiles under in vitro controlled conditions characterized. By testing different sucrose concentrations and the effects of two types of biotic elicitors, it was found that 3% (w/v) sucrose concentration in the liquid medium enhanced the production of hydrolytic enzymes. Moreover, casein hydrolysate at 0.5 and 1.5 g/L promoted protein production, whereas yeast extract (0.5 g/L) enhanced glycosidase activity. Meanwhile, chitosan (0.05 and 0.1 g/L) enhanced glycosidases, alkaline phosphates, and protease activities.

Bioprocess development for enhancing cell biomass, differential picrosides accumulation, and gene expression profiling at shake flask levels in suspension cultures of Picrorhiza kurroa

Plant cell suspension culture is an alternative and perpetual source for large-scale metabolites production in significantly less time. However, this approach has not yet been executed in Picrorhiza kurroa for their specialized metabolites production. Therefore, the present study aimed to develop the suspension culture process from the rhizome and leaf callus of P. kurroa by optimizing the physical cultivation parameters. Furthermore, the established cultures were subjected to sequential scale-up at 250, 500, 1000, 2000, and 5000 mL shake flask levels to evaluate growth, metabolites accumulation, and gene expression profiling of picrosides biosynthesis. Concurrently, the growth, metabolites content, and gene expression patterns were also assessed at the 4th, 8th, and 12th months of the incubation period. The investigation results revealed that the cell biomass in suspension cultures significantly improved solely by varying the culture parameters. The initial inoculum density (3 g/75 mL), subculture by pouring method, shaking speed (80 rpm), and pH (5.8) were suitable for enhanced production of cell biomass in rhizome and leaf suspension cultures in a 250 mL shake flask. During sequential scale-up, the cell biomass was reached in rhizome suspension (335 g FW; 13.80 g DW) and leaf suspension (263.33 g FW; 10.87 g DW) under optimized parameters at 5000 mL shake flask level. However, a maximum increase in the cell growth index and cell biomass (DW) in g/L of the medium was observed in a 250 mL flask compared to other shake flask volumes. In rhizome and leaf suspension, total picrosides (PI, PII, and PIII) (rhizome: 2.36–2.48 mg/g DW; leaf: 2.50–2.67 mg/g DW) and total precursors (vanillic acid, caffeic acid, cinnamic acid, catalpol, and aucubin) (rhizome: 1.35–1.37 mg/g DW; leaf: 1.20–1.45 mg/g DW) content range was observed during sequential scale-up at 250–5000 mL shake flask levels. Hence, no significant level of variation was observed in picrosides content (PI, PII, and PII in mg/g DW) with respect to different shake flask volumes (250–5000 mL) in rhizome and leaf suspension culture, respectively. Nevertheless, the productivity of metabolites content in mg/L of medium with respect to cell growth in g/L of medium showed a significant level of variation. The 250 mL shake flask volume culture of rhizome and leaf cell suspension accumulated the maximum total picrosides (31.07 and 31.20 mg/L of medium) and total precursors (17.04 and 16.94 mg/L of medium) content with cell biomass (12.53 and 11.69 g/L of medium), respectively. The results also revealed that the shikimate/phenylpropanoid pathway (EPSPS), mevalonate pathway (PMK and GDPS), non-mevalonate pathway (ISPD, DXPS, and ISPE), and iridoid pathway (GS, G10H, UGT, F3D, SQM, and ACT) genes showed upregulated expression in the rhizome and leaf suspension cultures. Analyzing the 1st month cultures with 4th, 8th, and 12th month cultures, the study depicted that growth index, cell viability, metabolites content, productivity in mg/L of the medium, and gene expression pattern were decreased with the age of cultures under one year of incubation. The present investigation delivered a simple yet feasible suspension culture process for metabolites enriched cell biomass production in P. kurroa.

Production of the SARS-CoV-2 Spike protein and its Receptor Binding Domain in plant cell suspension cultures

The COVID-19 pandemic, caused by the worldwide spread of SARS-CoV-2, has prompted the scientific community to rapidly develop efficient and specific diagnostics and therapeutics. A number of avenues have been explored, including the manufacture of COVID-related proteins to be used as reagents for diagnostics or treatment. The production of RBD and Spike proteins was previously achieved in eukaryotic cells, mainly mammalian cell cultures, while the production in microbial systems has been unsuccessful until now. Here we report the effective production of SARS-CoV-2 proteins in two plant model systems. We established transgenic tobacco BY-2 and Medicago truncatula A17 cell suspension cultures stably producing the full-length Spike and RBD recombinant proteins. For both proteins, various glycoforms were obtained, with higher yields in Medicago cultures than BY-2. This work highlights that RBD and Spike can be secreted into the culture medium, which will impact subsequent purification and downstream processing costs. Analysis of the culture media indicated the presence of the high molecular weight Spike protein of SARS-CoV-2. Although the production yields still need improvement to compete with mammalian systems, this is the first report showing that plant cell suspension cultures are able to produce the high molecular weight Spike protein. This finding strengthens the potential of plant cell cultures as production platforms for large complex proteins.

Chitosan nanoparticles and their combination with methyl jasmonate for the elicitation of phenolics and flavonoids in plant cell suspension cultures

Productivity enhancement approaches, such as elicitation can overcome the limitations of low metabolite(s) yield in in vitro plant cell culture platforms. Application of biotic/abiotic elicitors triggers molecular responses that lead to a concomitant enhancement in the production of metabolites. Nanoparticles have been tested as alternatives to commonly studied biotic/abiotic elicitors. However, most nanoparticles explored are of metallic origin, which raises concerns about their cytotoxicity, disposal post-elicitation, and may limit downstream applications of metabolites. Here, we report the synthesis and application of biopolymeric methyl jasmonate-loaded chitosan nanoparticles (MJ-CNPs) and empty CNPs (size <100 nm) as nano-elicitors, which were simple to synthesize, cost-effective and safe. Enzymatic and metabolic investigations revealed that MJ-CNPs and empty CNPs improve and prolong phenylalanine ammonia-lyase enzyme activity and production of phenolics and flavonoids. The data provides the first evidence of MJ-CNPs and empty CNPs as nano-elicitors that prolong the production of metabolites in plant cell suspension cultures.

Formation and analysis of structured solid foam patties based on crosslinked plant cell suspension cultures

Cellular agriculture is a new alternative food production technology that combines the cultivation of plant, animal or microbial cells with biotechnology. However, it can be challenging to manufacture cell-based products that reproduce the texture and organoleptic properties of conventional foods. Here, we developed a strategy to design the architecture of food products making use of plant cell suspension cultures derived from arctic bramble and rowan. Cellulose and pectin in the plant cell walls were cross-linked with citric acid and subject to partial alkaline hydrolysis under different conditions, followed by freeze drying in molds to form structured solid foam patties. We used a range of structural and mechanical characterization techniques and a trained sensory panel to show that highly nutritious plant cell cultures can form edible matrices with appropriate sensory qualities. The addition of citric acid without a base achieved a more acidic and berry-like flavor, whereas high doses of hydroxide and high temperatures conferred greater bite resistance. This new understanding of food structure design will facilitate the development of sustainable, healthy, and nutritious alternative food production technologies largely decoupled from conventional agriculture.

Plant Cell Suspension Culture: Modern Approaches and Problems in Drug Production (Review)

Plant Cell Suspension Culture: Modern Approaches and Problems in Drug Production (Review)

Highly Efficient Bioconversion of trans-Resveratrol to δ-Viniferin Using Conditioned Medium of Grapevine Callus Suspension Cultures.

δ-Viniferin is a resveratrol dimer that possesses potent antioxidant properties and has attracted attention as an ingredient for cosmetic and nutraceutical products. Enzymatic bioconversion and plant callus and cell suspension cultures can be used to produce stilbenes such as resveratrol and viniferin. Here, δ-viniferin was produced by bioconversion from trans-resveratrol using conditioned medium (CM) of grapevine (Vitis labruscana) callus suspension cultures. The CM converted trans-resveratrol to δ-viniferin immediately after addition of hydrogen peroxide (H2O2). Peroxidase activity and bioconversion efficiency in CM increased with increasing culture time. Optimized δ-viniferin production conditions were determined regarding H2O2 concentration, incubation time, temperature, and pH. Maximum bioconversion efficiency reached 64% under the optimized conditions (pH 6.0, 60 °C, 30 min incubation time, 6.8 mM H2O2). In addition, in vitro bioconversion of trans-resveratrol was investigated using CM of different callus suspension cultures, showing that addition of trans-resveratrol and H2O2 to the CM led to production of δ-viniferin via extracellular peroxidase-mediated oxidative coupling of two molecules of trans-resveratrol. We thus propose a simple and low-cost method of δ-viniferin production from trans-resveratrol using CM of plant callus suspension cultures, which may constitute an alternative approach for in vitro bioconversion of valuable molecules.

Plant Cell Wall Proteomes: The Core of Conserved Protein Families and the Case of Non-Canonical Proteins.

Plant cell wall proteins (CWPs) play critical roles during plant development and in response to stresses. Proteomics has revealed their great diversity. With nearly 1000 identified CWPs, the Arabidopsis thaliana cell wall proteome is the best described to date and it covers the main plant organs and cell suspension cultures. Other monocot and dicot plants have been studied as well as bryophytes, such as Physcomitrella patens and Marchantia polymorpha. Although these proteomes were obtained using various flowcharts, they can be searched for the presence of members of a given protein family. Thereby, a core cell wall proteome which does not pretend to be exhaustive, yet could be defined. It comprises: (i) glycoside hydrolases and pectin methyl esterases, (ii) class III peroxidases, (iii) Asp, Ser and Cys proteases, (iv) non-specific lipid transfer proteins, (v) fasciclin arabinogalactan proteins, (vi) purple acid phosphatases and (vii) thaumatins. All the conserved CWP families could represent a set of house-keeping CWPs critical for either the maintenance of the basic cell wall functions, allowing immediate response to environmental stresses or both. Besides, the presence of non-canonical proteins devoid of a predicted signal peptide in cell wall proteomes is discussed in relation to the possible existence of alternative secretion pathways.

Long-term cell suspension culture of Thymus persicus (Lamiaceae): A novel approach for the production of anti-cancer triterpenic acids

Triterpenic acids (TAs) such as betulinic acid (BA), oleanolic acid (OA), and ursolic acid (UA) are well-known natural compounds found in many plant taxa possessive a wide spectrum of biological activities and potential medicinal uses. Thymus persicus has been already reported as a rich source of TAs. Nowadays, long-term culture is an efficient technique in increasing biosynthesis and production of specialized metabolites in the plant cell suspension cultures with the possibility of stimulation, metabolic regulation, permeabilization, and up-scaling to bioreactors. In the present study, both batch and long-term cell suspension culture (CSC) of the plant were developed for the production of these compounds. A maximum specific cell growth rate of 5.7 g day−1, a doubling time of 42 h, and the highest cell viability (94.7%) throughout the growth period were measured. The highest cell fresh weight in CSC and its long-term culture was 100.63 ± 4.2 and 171.6 ± 4.1 g L−1 at day 21 and 35 of culture, respectively. A positive correlation between cell growth and the content of TAs in long-term culture was observed. The plant cells produced the highest levels of TAs in the mid-exponential phase and held their production stability during the long-term culture period. The highest content (mg 100 g−1 DW) of BA (163.11 ± 4.3) was measured in the second week of culture, while the maximum level of OA (10.22 ± 0.1) and UA (11.01 ± 0.4) was recorded in the third week of culture period. The present findings can be interestingly considered for further commercial production of valuable TAs at large-scale cultures in bioreactors.

Trends in the biotechnological production of isoflavonoids in plant cell suspension cultures

Trends in the biotechnological production of isoflavonoids in plant cell suspension cultures

Biomass Accumulation of Gynostemma Pentaphyllum (Thunb.) Makino in Cell Suspension Cultures inhibiting Human Cancer Cell Growth

Gynostemma pentaphyllum (Thunb.) Makino (GpM) is a medicinal plant in traditional medicine throughout Asia for the treatment of several diseases including cancer. GpM plant cell suspension cultures provide a time and cost effective well-controlled means promising a high-yielding biomass production of pharmaceutical compounds. The purpose of the current work is to investigate the effect of GpM cell suspension cultures on human cancer cell lines growth. The biomass was produced by cell suspension culture of GpM callus into 250 mL Erlenmeyer flask containing 50 mL of liquid medium culture. Gypenosides in GpM were confirmed by HPLC. Pharmacological activities of GpM extract were tested on human cancer cell line (HepG2, Huh7, A549 and HL-60) using Sulforhodamine B (SRB) cytotoxicity assay and Tetrazolium (MTT) assay. We successfully produced 5.485 ± 0.223 g GpM fresh biomass per 250 mL Erlenmeyer flask after 18 days culture. Total gypenosides and Rb1 in dry cell suspension were 48.844 ± 3.933 mg/g and 0.041 ± 0.004 mg/g. The crude extract from GpM cell suspension cultures exhibited significant cancerous cell growth inhibition in a dose dependent manner. From the MTT assay and SRB cytotoxicity assay, it is obvious that GpM cell suspension culture extract at 200 μg/mL significantly inhibited the growth of multiple human cancer cells including hepatoma cell lines (HepG2, Huh7), lung carcinoma cell line (A549) and leukemia cell line (HL-60). Anti-cancer cell proliferation properties of GpM cell suspension culture were significantly higher than those of natural plants’ extracts. In this framework, GpM in cell suspension cultures was found to inhibit the proliferation of several human cancer cells. Biomass accumulation of GpM in cell suspension cultures may contribute to the development of efficient strategies for plant-derived anticancer compound production.