Suspension Culture Medium Research Articles

Abstract 227: Novel and easy-to-use approach to triple-negative breast cancer tumoroid culture

Abstract Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer diagnoses in the United States. Patients with metastatic TNBC have a low survival rate, driven in part by limited treatment options for the disease. Tumoroids, also known as cancer organoids, have been shown to better maintain tumor-specific mutational and gene expression profiles over long-term culture, and evidence is growing that the functional response of tumoroids to therapeutics better recapitulates in vivo response. These improved preclinical models of TNBC may facilitate the development of new therapies, but tumoroids are yet to be widely adopted due to challenging culture methods. In this study, we show that two TNBC tumoroid models publicly available through the National Cancer Institute Patient-Derived Models Repository can be expanded in suspension culture using Gibco™ OncoPro™ Tumoroid Culture Medium supplemented with FGF10 and beta-estradiol. We compared the bulk tumoroid morphologies, growth rates, maintenance of mutational and gene expression profiles, and molecular phenotypes of cells grown in the NCI-recommended medium in embedded culture, and the OncoPro medium in embedded culture and suspension culture. We saw comparable morphologies and growth rates in all culture conditions. A multivariate analysis of the allelic frequencies of single nucleotide variants and ploidy values of copy number variants for cells expanded in all experimental conditions revealed >95% and >88% correlation, respectively, with the initial material. Interestingly, despite the change in the medium formulation, gene expression was also highly correlated between the initial material and the OncoPro medium conditions for >20,000 RefSeq genes (Pearson r>0.94) and for a cancer-gene specific panel of 1,423 genes (r>0.87). Further analysis showed unchanged expression levels in 97% of total genes analyzed and the basal molecular subtype was maintained for both lines in all conditions. The OncoPro media system was also used to derive a new TNBC tumoroid line, which demonstrates consistent growth rate and maintains the patient-specific mutational profile of the original breast tumor. Differential gene expression analysis revealed that changes in the gene expression profile were largely due to the loss of non-malignant cell populations from the original tumor. Finally, we utilized this new tumoroid line to examine donor-specific killing efficiencies of primary natural killer cells in a co-culture experiment. The discrepancy in the dynamics of killing a tumoroid line versus the standard K562 cell line indicates that tumoroids may offer an improved approach for screening new cell therapies. In summary, the easy-to-adopt OncoPro medium and culture method does not change the characteristics of existing models and may be used to derive new tumoroid lines, both of which can be leveraged for preclinical studies to develop critical new therapies for TNBC. Citation Format: Brittany Balhouse, Chris Yankaskas, Colin Paul, Shyanne Salen, Sylvia Beam, Pradip Shahi Thakuri, Matt Dallas, David Kuninger. Novel and easy-to-use approach to triple-negative breast cancer tumoroid culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 227.

In vitro effect of phytosynthesised AgNPs to enhance plantlets and biomass production in Nyctanthes arbor-tristis L.

This work was aimed to establish an effective approach for direct regeneration of Nyctanthes arbor-tristis L. using nodal segment. Amongst the different combinations of plant growth regulators (PGR's), the maximum number of shoots/explant (32.94 ± 0.86) with 3.14 ± 0.08 cm shoot length was attained on Murashige and Skoog (MS) medium fortified with 3.5 mg /L 6-benzyladenine (BA) and 0.3 mg/L concentration of 1-phenyl-3-(1, 2, 3- thiadiazol-5-yl)- urea (TDZ). Besides, nodal segments of Nyctanthes grown on MS medium were used to analyze the effect of biogenic AgNPs (0,2,4,6,8 and 10 mg/L) on plant growth & multiplication in solid & suspension culture medium. After 35 days of in vitro shoot proliferation, the shoot number, shoot length, chlorophyll content, fresh & dry matter, guaiacol peroxidase (GPOX) and catalases enzyme (CAT) activities of plants were quantified. The administration of AgNPs (6 mg/L) along with the lower concentration of BA (1 mg/L) could enhance shoot multiplication from (1.29 ± 0.14) to (25.17 ± 0.45), chlorophyll content as well as the GPOX, and CAT enzyme activities in suspension culture medium. Optimized rooting response (12.15 ± 0.63) was noted on the half-strength MS medium supplemented with 0.5 mg/L concentration of Indole acetic acid (IAA), The maximum in vitro raised plantlets was hardened & acclimatized in a ratio of 6:2:2:1(garden soil: cocopeat: vermiculite: pearlite) potting mixture. The regenerated plantlets showed 80% survival rate under greenhouse conditions. Out of twenty PCR-based RAPD (Random Amplified Polymorphic DNA) molecular markers, 8 primers produced a total of 350 scorable bands which showed 90–99% similarity with mother plant.

In vitro propagation and improving accumulation of coumarin in Lycium barbarum, a rare plant in the flora of Egypt

BackgroundThe conservation of natural populations of plants is very important for maintaining biological diversity. Lycium barbarum (goji berries) is classified as a very rare plant in the Egyptian flora. Goji berry is a fruit commonly known as ‘super-fruit,’ due to its beneficial constituents and medicinal properties. The aim of this study was to determine the influence of media formula added with various cytokinins on axillary bud induction of L. barbarum from shoot tip. An additional objective of this study was to investigate the effect of 2,4-D, TDZ, zeatin, IBA and NAA on growth and development of the plant through different stages. The present study also aims to improve coumarin accumulation as one of the most active constituents in the plant.ResultsSterilized shoot tips of L. barbarum were cultured without growth regulators on different media formulas: Murashige and Skoog basal medium (MS medium), Gamborg’s medium (B5 medium) and Woody plant medium (WP medium). MS medium gave the best shoot induction. The maximum multiplication rate of shoots was recorded on MS medium supplemented with 225.24 µM 6-benzyl amino purine (BAP). The best callus induction rate from in vitro derived young leaves was obtained on MS medium with 552.60 µM thidiazuron (TDZ). At 176.20 µM TDZ, the maximum shoot proliferation ability was obtained through organogenesis from the callus. MS medium with 220.25 µM TDZ produced the highest number (15 buds) and forming rate (96%) of adventitious buds through direct organogenesis. On MS medium with 304.86 µM naphthalene acetic acid (NAA), 100% rooting was obtained. Almost 95% of the in vitro-generated plantlets were successfully acclimatized. Furthermore, suspension culture containing MS medium with 552.60 µM TDZ and cinnamic acid at a concentration of 763 μM increased coumarin synthesis (1.95-fold), compared to control.ConclusionThe in vitro regeneration of plants via leaf-mediated shoot organogenesis protocol presented in this study is well suited for L. barbarum propagation and produced large quantities of identical planting materials for desert revegetation and pharmaceutical industries. A very effective indirect regeneration method via callus culture provides a potential for this ethnomedicinal plant to be improved by genetic transformation procedures, in addition to mass propagation of elite plants. The cell suspension culture medium with 763 µM cinnamic acid had the highest coumarin accumulation.

Metabolic Alteration Analysis of Steroid Hormones in Niemann-Pick Disease Type C Model Cell Using Liquid Chromatography/Tandem Mass Spectrometry.

Niemann–Pick disease type C (NPC) is an autosomal recessive disease caused by a functional deficiency of cholesterol-transporting proteins in lysosomes, and exhibits various clinical symptoms. Since mitochondrial dysfunction in NPC has recently been reported, cholesterol catabolism to steroid hormones may consequently be impaired. In this study, we developed a comprehensive steroid hormone analysis method using liquid chromatography/tandem mass spectrometry (LC–MS/MS) and applied it to analyze changes in steroid hormone concentrations in NPC model cells. We investigated the analytical conditions for simultaneous LC–MS/MS analysis, which could be readily separated from each other and showed good reproducibility. The NPC phenotype was verified as an NPC model with mitochondrial abnormalities using filipin staining and organelle morphology observations. Steroid hormones in the cell suspension and cell culture medium were also analyzed. Steroid hormone analysis indicated that the levels of six steroid hormones were significantly decreased in the NPC model cell and culture medium compared to those in the wild-type cell and culture medium. These results indicate that some steroid hormones change during NPC pathophysiology and this change is accompanied by mitochondrial abnormalities.

Production of recombinant human acid α-glucosidase with mannosidic N-glycans in α-mannosidase I mutant rice cell suspension culture

High-mannose glycans, containing 6–9 mannose residues, are favorable for in vitro mannose phosphorylation to produce mannose-6-phosphate (M6P) residues, which are essential for uptake into target cells during enzyme replacement therapy of Pompe disease. Golgi α-mannosidase-I mediates mannose trimming in the N-glycosylation pathway. In this study, mutant rice with a T-DNA insertion in the Os04g0606400 (∆α-manI) gene was used to produce recombinant human acid α-glucosidase (rhGAA). Expressed and secreted rhGAA from ∆α-manI (∆α-manI-GAA) in suspension culture media was purified, and analysed by MALDI-TOF. It showed the presence of high-mannose type N-glycans with Man8 (63.8%), Man7 (22.5%), and Man6 (5.4%).

Plant tissue culture of cat whiskers (Orthosiphon aristatus Blume Miq): A review of secondary metabolite production and micropropagation

Background: The cat whiskers plant (Orthosiphon aristatus Blume Miq) is widely used as a raw material in traditional medicine for one of its many properties, i.e. antiviral activity. Three varieties of cat whiskers grow in Indonesia, classified according to the colour of their flower: white, white-purple, and purple. The purple variety of cat whiskers is endangered, so it is necessary to propagate this plant. Objectives: This research aimed to obtain appropriate protocols for plant propagation and production of active compounds of cat whiskers by in vitro culture. Methods: Data were collected from various online journal sites such as PubMed, ResearchGate, and Scopus. This review discusses several aspects, including callus induction efforts, modification of cell suspension cultures, and efforts to propagate the cat whiskers plant by in vitro culture. Results: Callus induction of white cat whiskers with purple and purple hues could take place on Murashige and Skoog (MS) media added with growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D) 0.4 mg/L. Suspension culture medium (MS+ 2,4-D 1 mg/L + NAA 1 mg/L) can increase cell biomass and rosmarinic acid levels. Media MS + 6-benzyl amino purine (BAP) 3 mg/L + 1-naphthaleneacetic acid (NAA) 2 mg/L can induce the growth of shoots of white cat whiskers with purple and purple patterns. Root induction of the two varieties of cat whiskers could take place on MS + Indole-3-butyric acid (IBA) 0.75 mg/L medium. Conclusion: Efforts to produce secondary metabolites and plant propagation of cat whiskers by in vitro culture have been successful. It is necessary to enhance the production and propagation using bioreactors to yield more active compounds and raw materials of the cat whiskers plant.

Plant regeneration via protoplast electrofusion in cassava

Protoplast electrofusion between callus protoplasts of cultivar TMS60444 and mesophyll protoplasts of cultivar SC8 was performed as an approach for the genetic improvement of cassava. The fusion products were subsequently cultured in protoplast culture medium (TM2G) with gradual dilution for approximately 1–2 months. Then the protoplast-derived compact calli were transferred to suspension culture medium (SH) for suspension culture. The cultured products developed successively into embryos, mature embryos, and shoots on somatic embryo emerging medium (MSN), embryo maturation medium (CMM), and shoot elongation medium (CEM), respectively. And the shoots were then rooted on Murashige and Skoog (1962) medium (MS). Sixty-six cell lines were obtained and 12 of them developed into plantlets. Based on assessment of ploidy level and chromosome counting, four of these plantlets were tetraploid and the remaining eight were diploid. Based on assessment of ploidy level and simple sequence repeat (SSR) analysis, nine tetraploid cell lines, one diploid variant plant line and nine variant cell lines were obtained. The diploid variant plant line and the nine variant cell lines all showed partial loss of genetic material compared to that of the parent TMS60444, based on SSR patterns. These results showed that some new germplasm of cassava were created. In this study, a protocol for protoplast electrofusion was developed and validated. Another important conclusion from this work is the confirmation of a viable protocol for the regeneration of plants from cassava protoplasts. Going forward, we hope to provide technical guidance for cassava tissue culture, and also provide some useful inspiration and reference for further genetic improvement of cassava.

Molecular identification of a Bacillus cereus strain from Murrah buffalo milk showed in vitro bioremediation properties on selective heavy metals.

Objective:This study aims for molecular identification of naturally growing Bacillus cereus strain from a unique source, able to survive, and alleviate heavy metals from the nature.Materials and Methods:Pure isolate from Murrah buffalo milk was prepared in B. cereus selective Polymyxin pyruvate egg-yolk mannitol–bromothymol blue agar (PEMBA) medium through a cascade of contamination free subcultures. The morphological and biochemical tests were done prior to 16S rRNA gene sequencing for strain identification and further physiological tests. The test strain was inoculated in both solid and suspension culture medium supplemented individually with Cd, Cu, Ag, and Zn to reveal the qualitative and quantitative heavy metal tolerance properties, respectively. Finally, the data collected from the in vitro assessment was statistically analyzedResults:Molecular analysis revealed that the test strain was B. cereus BF2, which was motile, catalase positive and Gram positive rod. B. cereus BF2 was found significant at 0.3% bile salt tolerance [two-way analysis of variance (ANOVA)—p value is < 0.0001] where, t-test p value is < 0.0002 between Control Group (CG) and TGR-1; p < 0.037 between TGR-1 and 2; p < 0.0014 between CG and TGR-2. Similarly, B. cereus BF2 was significant in pH tolerant up to 8.0 with p < 0.0115 (in scale p < 0.05). The heavy metal tolerance test revealed that the test metals could not stop the growth of B. cereus BF2 even after 24 h of incubation but partially suppressed the growth kinetics for letting into stationary phase. Among the four heavy metals, Cd and Zn showed partial antagonism to the growth of B. cereus BF2. The survivability was highly significant in the medium supplemented with Zn (p < 0.0001) and Ag (p < 0.018).Conclusion:Bacillus cereus BF2 can survive in selective heavy metals with metal resistance and biodegradation capacity.

Isolation of cancer stem cells and the establishment of a H 2O 2-resistant cancer stem cell model

To isolate cancer stem cells (CST) from human breast cancer cell line (MCF-7) and study their sensitivity toward oxidative stress. MCF-7 cells were cultured in serum-free suspension culture medium to identify cells forming the sphere phenotype. The morphological changes of MCF-7 cells were observed by inverted phase contrast microscope (compared with MCF-7 cells cultured in serum-free suspension culture medium). The expression of CST marker CD133 was detected by immunocytochemical staining in CST cell spheres (experimental group) with a diameter of 100 μm and MCF-7 cells (control group) with a fusion degree of 70%. The positive rate of CD133 was detected by flow cytometry in the third generation of tumor cells with diameter of 150 μm. The second generation of tumor globular cells (experimental group) with diameter of 150 μm and corresponding MCF-7 cells (control group) were taken to be damaged by 50 mol/L H 2O 2 for 120 minutes. The expression of DNA damage marker histone H2AX phosphorylation (γH2AX) was detected by immunocytochemical staining. Inverted phase contrast microscopy showed that MCF-7 cells grew initially in a single-cell adherent state, then aggregated and grew in serum-free suspension culture medium, and finally formed CST cell spheres, while the control MCF-7 cells cultured in MCF-7 cell culture medium grew extensively and could not grow in suspension. Fluorescence microscopy showed that the expression of CD133 in MCF-7 cells of control group was negative, while that in experimental group was positive. Flow cytometry showed that CD133 was positive in CST cells, and the positive rate was 92%. Inverted fluorescence microscopy showed that the expression of γH2AX in CST tumor spheres of experimental group was significantly lower than that in MCF-7 cells of control group after 120 minutes of H 2O 2 injury. Serum-free suspension culture medium can produce globular CST cells from MCF-7 tumor cell line, which have strong antioxidant damage.

Purified Anthocyanin from in vitro Culture of Bridelia retusa (L.) Spreng. Capable of Inhibiting the Growth of Human Oral Squamous Cell Carcinoma Cells

The present study aims in vitro cell suspension culture of Bridelia retusa, isolation of anthocyanin, purification, fractionation and its anti-metastatic potential against oral squamous carcinoma cells. Experimental results reveal that 2, 4-D either alone or in combination with kinetin supplemented in MS medium showed significant initiation of callus from leaf explants than stem. Growth hormones, pH, light, and carbon source influence anthocyanin synthesis. Maximum callus induction was noticed with 2.5 mg/L N6-benzyladenine (BA) + 2 mg/L 2, 4-dichlorophenoxyacetic acid (2, 4-D) (98.9%). Fresh and dry weight of the calli were i.e., 1.9 ± 0.04 and 0.45 ± 0. 03 g respectively. Optimal response was seen with light on MS medium contain 4% glucose + 2.5 mg/L BA and 2 mg/L 2, 4-D at pH 3.5 yielded 2.8 mg /g of anthocyanins. Suspension culture medium fortified with 2, 4-D (2.5 mg/L) + BA (2 mg/L) at pH 5.0 induced anthocyanin production at pH 4.4 – 4.6. HCl-ethanol extraction for 90 min yielded the maximum anthocyanin content. Fractionation of anthocyanin using HPLC coupled with mass spectrometry revealed 07 fractions such as acylated cyanidins, two peonidins, cyanidin 3-p-coumaroyl and feruloyl diglucoside-5-glucosides. In the search of novel therapeutic drugs against cancer, cytotoxicity effect of B.retusa anthocyanin extracts on human oral squamous cell carcinoma (SCC4, SCC9 and SCC25) cells using cell adhesion and cell viability assay was carried. The morphological alterations in SCCs cells after treatment with B.retusa anthocyanin includes nuclear condensation, fragmentation and apoptotic cells as revealed by Hoechst stain. Flow cytometry showed arresting of SCC25 cells mostly in the G0/G1 and S-G2/M stages with a concomitant up regulation of sub-G1 fraction, indicating cell death by apoptosis. Apoptosis was further substantiated by the activation of caspase-3 expression in the SCC25 cells treated with B.retusa anthocyanin. Thus, it is possible to suggest that B.retusa anthocyanin cause apoptosis of SCCs and warrant further investigation using animal models.

β-Ecdysterone accumulation and regulation in Ajuga multiflora Bunge suspension culture.

Ajuga multiflora Bunge cells contain β-ecdysterone (β-EC) that regulates the molting process of insect larvae. In this study, different conditions of culture have been studied to optimize the production of β-EC. A. multiflora Bunge growth fitted the curve of logistic equation with one growth cycle of 17days. The electric conductivity of medium had a negative correlation with not only the weight of dry cell but also the β-EC accumulation, and thus, could be used for monitoring the peak of both cell growth and β-EC accumulation. The pH value of the culture medium varied from 4.67 to 5.84 and reached the maximum at the end of the culture (on the 17th day). The relation of cell growth and nutrient consumption in A. multiflora Bunge cell suspension culture was distinctly correlated. Continuous subculture caused a reduction in β-EC synthesis; passages 7-15, the β-EC content declined (p<0.05). At passage 11, the β-EC content was only 42.72% of that at passage 5. Additives such as mevalonic acid (MVA), l-phenylalanine (l-Phe), α-pinene, terpineol, and nitric oxide (NO) in the suspension culture medium, could significantly promote the cell growth and stimulate β-EC accumulation. The optimal concentrations of l-Phe, MVA, terpineol, and α-pinene were 0.2mmol/l, 10mg/l, 1mmol/l and 6mmol/l, respectively, with the β-EC concentrations as 1.914±0.1948mg/g (p<0.01), 6.012±0.4252mg/g (p<0.01), 5.147±0.4819mg/g (p<0.01), 2.801±0.1253mg/g (p<0.01), respectively. The optimal concentration of sodium nitroprusside, the provider of NO, was 3mmol/l with the β-EC concentration 2.87±0.2493mg/g (p<0.01). The results offer a strategy for massive production of β-EC.

Optimization of culture conditions for the production of polysaccharides and kinsenoside from the rhizome cultures of Anoectochilus roxburghii (Wall.) Lindl.

The present study designed two sets of experiments by using the uniform design method and investigated the effects of medium components on the accumulation of bioactive compounds (polysaccharide and kinsenoside) in rhizomes, in order to select a suitable culture medium for the rhizome suspension culture of Anoectochilus roxburghii (Wall.) Lindl. Among the combinations of Murashige and Skoog (MS) medium strengths and plant growth regulator (benzylaminopurine, BA; kinetin, KT; and α-naphthaleneacetic acid, NAA) concentrations, and the combinations of nitrogen, phosphorus, and sucrose concentrations, the maximum yield of polysaccharides and kinsenoside was achieved with 0.75 × MS + 2.0 mg L−1 BA + 0.2 mg L−1 KT + 0.5 mg L−1 NAA and 45 mM nitrogen + 0.93 mM phosphorus + 35 g L−1 sucrose, respectively. Therefore, the optimal rhizome suspension culture medium was 0.75 × MS medium supplemented with 2.0 mg L−1 BA, 0.2 mg L−1 KT, 0.5 mg L−1 NAA, and 35 g L−1 sucrose. Yeast extract (YE) enhanced bioactive compound accumulation in rhizomes. The polysaccharide and kinsenoside production was significantly improved when 75 mg L−1 YE was added to the culture medium after 30 d of rhizome suspension culture; 8.3 g L−1 of polysaccharide and 6.1 g L−1 of kinsenoside were obtained after 4 d of YE treatment. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of YE-treated rhizomes was higher than that of YE-untreated rhizomes, demonstrating enhanced antioxidant activity of the treated bioreactor-cultured rhizomes.

High yield production of folates from soybean callus cultures in response to elicitors.

Soybean (Indian variety, JS 335) callus growth and its folate content was sustained on Murashige and Skoog (MS) medium supplemented with optimized levels of auxins and cytokinins. Callus cultures exhibited moderate production of total folate. Callus growth was stimulated with highest biomass content of 21.3g/L DW (24days culture) on medium supplemented with 1.5mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.1mg/L Kinetin (Kn). Total folate production was enhanced by an increase in indole-3-acetic acid (IAA) from 0.5 to 1.0mg/L and decreased thereafter up to 1.5mg/L. 2,4-D at 1.5mg/L repressed the callus growth and also folate production. A concentration of 1.0mg/L IAA and 0.2mg/L Kn elicited folate production to a maximum total folate content of 323.82µg/100g DW. The results indicate that optimized medium for callus suspension cultures conceivably are applied for scale-up studies in bioreactors.

Establishment of Callus Induction and Cell Suspension Cultures of Dendrathema Indicum var. Aromaticum a Scented Chrysanthemum

Dendranthema indicum var. aromaticum is an important aroma plant in genus Dendrathema, and the establishment of callus cultures and cell suspension cultures is the basement of further protoplast fusion studies, which make it possible to breed new fragrant chrysanthemum. In this study, the effects of different plant growth regulating substances in different concentrations on callus induction were investigated with stem segments, leaves, petioles as explants. The results showed that the optimal explants were lower stem segments according to the percentage of callus formation, callus hardness, growth potential and shoot differentiation. The optimal induction mediums were MS supplemented with 1.0 mg．l-12.4 D and 0.2 mg．l-1 6-BA. The cell suspension culture system was established by using the subculture calli. The results showed that the suitable inoculum size was 2g and the suitable cell suspension culture medium was MS supplemented with 0.2 mg．l-1 6-BA and 0.5 mg．l-1 2,4-D. The time course of cell growth showed that the greatest cell fresh weight appeared on day 14 and the highest cell viability on day 3.

Fluid shear stress induces cancer stem cell-like phenotype in MCF7 breast cancer cell line without inducing epithelial to mesenchymal transition.

Metastasis is the leading cause of cancer deaths due to the spread of cancer cells through the blood vessels and the subsequent formation of secondary tumors. Metastasizing cancer cells in the human vasculature are called circulating tumor cells (CTCs) and are characterized to express the epithelial cell adhesion molecule (EpCAM). They are further known to survive physiological fluid shear stress (FSS) conditions. However, the effect of FSS on CTC molecular phenotype, such as the epithelial to mesenchymal transition (EMT) and cancer stem cell (CSC) expression, has not been extensively studied. Here, CTCs in FSS are evaluated in an invitro model system. MCF7 and MDA-MB-231 breast cancer cell lines were grown in adherent and suspension culture media. The cell lines were tested for EMT and CSC genetic and protein markers using qRT-PCR and flow cytometry, respectively. Suspension cells showed a significantly increased EMT signature compared to adherent cells (p<0.05), suggesting that they model cells detaching from primary tumors invivo. Upon application of FSS, MCF7 and MDA-MB-231 cells did not show a significant change in EMT expression (p>0.05), but there was a statistically significant increase of the CSC population in MCF7 suspension cultures (p<0.05). These results with MCF7 suggest that CTCs can be modeled invitro as non-adherent cancer cells in FSS and that they show an increased CSC-like signature during circulation, providing new insights to the importance of CSC-targeting strategies when treating metastatic patients.

Culture medium refinement by dialysis for the expansion of human induced pluripotent stem cells in suspension culture

Human induced pluripotent stem cells (hiPSCs) secrete essential autocrine factors that are removed along with toxic metabolites when the growth medium is exchanged daily. In this study, after determining the minimum inhibitory level of lactic acid for hiPSCs, a medium refining system was constructed by which toxic metabolites were removed from used culture medium and autocrine factors as well as other growth factors were recycled. Specifically, about 87% of the basic fibroblast growth factor and 80% of transforming growth factor beta 1 were retained in the refined medium after dialysis. The refined medium efficiently potentiated the proliferation of hiPS cells in adherent culture. When the refining system was used to refresh medium in suspension culture, a final cell density of (1.1±0.1)×106cellsmL-1 was obtained, with 99.5±0.2% OCT 3/4 and 78.3±1.1% TRA-1-60 expression, on day 4 of culture. These levels of expression were similar to those observed in the conventional suspension culture. With this method, culture medium refinement by dialysis was established to remove toxic metabolites, recycle autocrine factors as well as other growth factors, and reduce the use of macromolecules for the expansion of hiPSCs in suspension culture.

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.

Isoflavone Augmentation in Soybean Cell Cultures Is Optimized Using Response Surface Methodology

Glycine max contains potential therapeutic isoflavones, and its productivity in plants is considerably influenced worldwide by several biotic and abiotic factors. Optimization of soybean cell suspension cultures (Indian variety, JS 335) to maximize the cell growth and isoflavone production in the present study was performed using response surface methodology (RSM) with three independent variables of plant growth regulators, 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphthalene acetic acid (α-NAA), and kinetin (Kn). The maximum biomass achieved was 70.62 g/L dry weight (dw) using the optimized medium of 2.10 mg/L 2,4-D, 5.52 mg/L α-NAA, and 0.35 mg/L Kn supplemented in the Murashige and Skoog (MS) basal medium. The total isoflavone content of 38.59 mg/g of dw was obtained in the medium with optimized conditions of 1.33 mg/L 2,4-D, 1.76 mg/L α-NAA, and 0.15 mg/L Kn. In comparison to field-grown soybean seeds, the cell suspension cultures profoundly augmented isoflavone concentrations. The optimized conditions for both biomass and total isoflavone content were evaluated by superimposing the contour plots. The results suggested that the optimized medium of cell suspension cultures possibly be used for scale-up studies in bioreactors to offer a high content of bioactive isoflavones.

Comparative study of the spinal tuberculosis model with the New Zealand rabbit

Objective To establish and evaluate a rabbit model of spinal tuberculosis.Methods Sixty-two New Zealand rabbits were randomly divided into 4 groups:experimental A group (sensitized by complete Freund's adjuvant,n=20),experimental B group (not sensitized by complete Freund's adjuvant,n=20),control group (n=20) and blank group (n=2).After experimental A group was sensitized by complete Freund's adjuvant,a hole was drilled beneath the upper endplate of the L4 vertebral body and filled with a gelfoam sponge,which was infused with 0.l ml H37Rv standard Mycobacterium tuberculosis suspension.Experimental B group was filled with a gelfoam sponge infused with 0.1 ml H37Rv standard Mycobacterium tuberculosis suspension (culture medium and saline in control group).The blank group received no treatment.Results The survival rate of postoperative 8 w was 89.5％,60.0％,94.7％ and 100％ in the experimental A,experimental B,control and blank group respectively.The model was successfully established in all surviving rabbits of the experimental A group.The vertebral body destruction of the experimental A group was 50％ by X-ray and 83.3％ by CT reconstruction and MRI at 4 w,while it was 58.8％ by X-ray and 100％ by CT reconstruction and MRI at 8 w.All surviving rabbits of the experimental A group had vertebral destruction,granulation and necrosis while 17.6％ had psoas abscess at 8 w.Histopathology study revealed numerous lymphocytes and epithelioid cells,trabecular bone fracture and coagulative necrosis in the vertebrae of the experimental A group.The bacterium culture was 52.9％ positive.There were 75％ vertebrae damage and 100％ psoas abscess in experimental B group.Nither control nor blank group showed such changes.Conclusion A New Zealand rabbit of spinal tuberculosis model can be successfully established by drilling a hole in the upper endplate of the vertebral body,filling with gelfoam sponge infused with H37Rv standard Mycobacterium tuberculosis suspension after sensitized by complete Freund's adjuvant. Key words: Tuberculosis, Spinal; Models, Animal; Mycobacterium tuberculosis; Freund's adjuvant; Rabbits

Optimization of BY‐2 cell suspension culture medium for the production of a human antibody using a combination of fractional factorial designs and the response surface method

We have developed a strategy for the optimization of plant cell suspension culture media using a combination of fractional factorial designs (FFDs) and response surface methodology (RSM). This sequential approach was applied to transformed tobacco BY-2 cells secreting a human antibody (M12) into the culture medium, in an effort to maximize yields. We found that the nutrients KNO₃, NH₄NO₃ and CaCl₂ and the hormones 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP) had the most significant impact on antibody accumulation. The factorial screening revealed strong interactions within the nutrients group (KNO₃, NH₄NO₃ and CaCl₂) and also individually between 2,4-D and three other components (KNO₃, NH₄NO₃ and BAP). The RSM design resulted in a fivefold increase in the antibody concentration after 5 days and a twofold reduction in the packed cell volume (PCV). Longer cultivation in the optimized medium led to the further accumulation of antibody M12 in the culture medium (up to 107 μg/mL, day 10). Because the packed cell volume was reduced in the optimized medium, this enhanced the overall yield by 20-fold (day 7) and 31-fold (day 10) compared to the conventional MS medium.