Tissue Culture Bags Research Articles

Competitive Growth Assay of Mutagenized Chlamydomonas reinhardtii Compatible With the International Space Station Veggie Plant Growth Chamber.

A biological life support system for spaceflight would capture carbon dioxide waste produced by living and working in space to generate useful organic compounds. Photosynthesis is the primary mechanism to fix carbon into organic molecules. Microalgae are highly efficient at converting light, water, and carbon dioxide into biomass, particularly under limiting, artificial light conditions that are a necessity in space photosynthetic production. Although there is great promise in developing algae for chemical or food production in space, most spaceflight algae growth studies have been conducted on solid agar-media to avoid handling liquids in microgravity. Here we report that breathable plastic tissue culture bags can support robust growth of Chlamydomonas reinhardtii in the Veggie plant growth chamber, which is used on the International Space Station (ISS) to grow terrestrial plants. Live cultures can be stored for at least 1 month in the bags at room temperature. The gene set required for growth in these photobioreactors was tested using a competitive growth assay with mutations induced by short-wave ultraviolet light (UVC) mutagenesis in either wild-type (CC-5082) or cw15 mutant (CC-1883) strains at the start of the assay. Genome sequencing identified UVC-induced mutations, which were enriched for transversions and non-synonymous mutations relative to natural variants among laboratory strains. Genes with mutations indicating positive selection were enriched for information processing genes related to DNA repair, RNA processing, translation, cytoskeletal motors, kinases, and ABC transporters. These data suggest that modification of DNA repair, signal transduction, and metabolite transport may be needed to improve growth rates in this spaceflight production system.

High-Content CD14+ Cells in Apheresis Products negatively affects the Transduction and Expansion of CAR T cells during Large-scale cGMP Manufacturing

Abstract Adoptive transfer of chimeric antigen receptor (CAR) engineered T cells is a promising strategy to treat patients with cancer. Apheresis products are commonly used as starting material for large-scale T cell selection using CD3/28 magnetic beads. At the time of collection, the composition in various cell lineages is highly variable. We have encountered manufacturing challenges with apheresis products containing high proportion of CD14+ cells. As an illustration, we investigated the transduction and expansion of T cells using an apheresis product derived from a patient with ALL and containing 70% CD14+ cells. 350 × 106 CD3+ cells were selected from a cryopreserved apheresis product using CD3/28 magnetic beads. A short adherence procedure decreased the CD14+ cell composition from 70% to 45%. On day 3, 350×106 vs 20 ×106 total cells were recovered from each group without and with the depletion step, respectively. Selected cells from both groups were transduced using the same dilution of vector stocks at a cell concentration of either 0.4×106/mL or 0.2×106/mL in tissue culture bags. We observed a drastic difference in transduction efficiency between the group without depletion (4.5%) and the group in which the CD14+ cells were depleted (38%). Cells from the depleted group expanded 350 fold from day 3 to day 10 (100% CD3+). Our results suggest that high CD14+ cell content poses a manufacturing challenge when the selection of the CD3+ cells in the apheresis product is performed with CD3/CD28 magnetic beads. The CD14+ cell threshold for successful manufacturing without depletion will be discussed and additional samples will be analyzed.

80. Depletion of High-Content CD14+ Cells from Apheresis Products is Critical for the Successful Transduction and Expansion of CAR T Cells During Large-Scale cGMP Manufacturing

Adoptive transfer of chimeric antigen receptor (CAR) engineered T cells holds great promise as a novel strategy to treat patients with cancer. Apheresis products are a major source of starting material for large-scale T cell selection using CD3/28 magnetic beads. At the time of collection, the composition in various cell lineages is highly variable. We have successfully manufactured CAR T cell products starting from apheresis products containing as few as 0.5% to 3% of CD3+ cells. However, we have experienced manufacturing challenges with apheresis products containing a high fraction of CD14+ cells. We conducted a study with an apheresis product derived from a patient with acute lymphoblastic leukemia containing 70% CD14+ cells. 350 × 106 CD3+ cells were selected from the cryopreserved apheresis product using CD3/28 magnetic beads either with or without a prior 1.5 hr monocyte/granulocyte adherence step. This short adherence procedure decreased the CD14+ cell composition from 70% to 45%. A second overnight depletion by adherence in T-flasks and re-stimulation with CD3/28 magnetic beads on day 1 were also performed in the depleted group to ensure the proper activation of CD3+ cells. On day 3, 350×106 vs 20 x106 total cells were recovered from each group without and with the depletion step, respectively. Selected cells from both groups were transduced using the same dilution of vector stocks at a cell concentration of either 0.4×106/mL or 0.2×106/mL in tissue culture bags. On day 7, we observed a drastic difference in transduction efficiency between the group without depletion (4.5%) and the group in which the CD14+ cells were depleted (38%). Moreover, starting with 108 × 106 selected cells on day 3, a mere total of 12.3 × 106 (14.8% CD3+) were recovered on day 11 in the group without depletion. By contrast, cells from the depleted group expanded 350 fold from day 3 to day 10 (100% CD3+). Our results suggest that high CD14+ cell content poses a manufacturing challenge when the selection of the CD3+ cells in the apheresis product is performed with CD3/CD28 magnetic beads. The CD14+ cell threshold for successful manufacturing without depletion remains to be determined. However, monitoring the CD14 cell content and depleting these cells from starting apheresis products with high CD14 content prior to CD3/CD28 magnetic bead selection are critical to ensure success of the CAR T cell manufacturing process.

Modificaciones al envase y al medio aumentan el intervalo de transferencia in vitro de plántulas de Musa spp.

Los bananos y plátanos (Musa spp.) son de los cultivos alimenticios más importantes del mundo. El USDA-ARS TARS mantiene una colección de alrededor de 164 accesiones de Musa spp. en el campo y almacenado in vitro. Las accesiones que se mantienen in vitro requieren transferencias rutinarias al absorberse los nutrientes. Los intervalos de transferencia se hacen, en promedio, cada seis meses en un proceso que requiere muchos recursos. Para extender el intervalo entre transferencias, se evaluaron tres modificaciones al medio nutritivo y tres envases de almacenamiento utilizando cuatro accesiones de Musa spp. Los envases de almacenamiento consistieron en tubos de cristal no sellados y sellados con Parafilm®, y bolsas de cultivo plásticas. Las tres modificaciones al medio nutritivo Murashige and Skoog (MS) fueron: concentración completa, % de concentración y concentración completa con 4% D-mannitol. Los efectos de los tratamientos se evaluaron mensualmente midiendo la apariencia general de la plántula, enraizamiento, y número de hojas y tallos. Para los tres diferentes medios y para las cuatro accesiones se vieron diferencias significativas en intervalos de transferencia para las plántulas en los tubos de cristal sellados con Parafilm® y en las bolsas de cultivo plásticas. El tratamiento de MS a la % de concentración retardó el crecimiento de las plántulas y mostró el tiempo más corto en almacenamiento para las cuatro accesiones independientemente del envase. El tiempo de almacenamiento se pudo prolongar a 12 meses con las bolsas de cultivo plásticas, y a 16 meses con los tubos sellados. Sin embargo, las bolsas de cultivo plásticas son más económicamente ventajosas debido a su simplicidad para propósitos de distribución y por su baja incidencia de contaminación.

Medium, container and genotype all influence in vitro cold storage of apple germplasm

The goal of this study was to evaluate the in vitro storage of apple germplasm by screening a range of genotypes followed by more comprehensive testing of multiple parameters on two genotypes of differing species, Malus domestica cultivar Grushovka Vernenskaya and wild Malus sieversii selection TM-6. Stored plants were rated on a 6 point scale (0 low to 5 high) for plant appearance at 3 month intervals after storage at 4°C. Combinations of carbon source (sucrose and/or mannitol), nitrate nitrogen content (25, 50 or 100%) and plant growth regulators (ABA, BAP, IBA) were studied in three types of containers (tissue culture bags, test tubes or jars). An initial screen of 16 genotypes stored in tissue culture bags indicated that plantlets could be stored at 4°C for 9–14 months without subculture on standard 3% sucrose Murashige and Skoog (1962) (MS) medium with no plant growth regulators (PGRs). In subsequent in-depth studies on the two genotypes, ANOVA indicated highly significant interactions of medium, container and genotype. ‘Grushovka Vernenskaya’ shoots with no PGRs and 3% sucrose remained viable (ratings of ≥1) for 21 months of storage in bags. Storage on reduced nitrogen (MS with 25% nitrogen), PGRs, and 3% sucrose kept ‘Grushovka Vernenskaya’ shoot condition rated >2 at 21 months. Addition of 0.5 or 1 mg−1 abscisic acid (ABA) also improved plant ratings at 21 months. The longest storage for ‘Grushovka Vernenskaya’ was 33–39 months with PGRs and 3% sucrose in either tubes or jars. Addition of abscisic acid (ABA) to the medium did not improve storage of plantlets in jars and tubes at 15 months. TM-6 stored best in tubes on 3% sucrose with PGRs or in jars on 2% mannitol and 2% sucrose. Overall it appears that cold storage of apple shoot cultures can be successful for 21 months in tissue culture bags with 25% MS nitrate nitrogen, 3% sucrose, and no PGRs or for 33 months in jars or tubes on MS with 3% sucrose and PGRs. Preliminary RAPD analysis found no significant differences between plants stored for 39 months and non-stored controls.

Non-destructive evaluation of in vitro-stored plants: A comparison of visual and image analysis

In vitro plants, in slow-growth storage require routine evaluation for assessment of viability and need for repropagation. Determination of plantlet health by visual assessment is subjective and varies by genus due to variations in growth pattern and plant structure. Developing a standardized plant evaluation system would improve the efficiency of in vitro storage. This study was initiated to develop digital image analysis techniques for plantlets during slow-growth cold storage and to compare that system with visual examinations. Pear (Pyrus communis L) cultivars were chosen for this initial trial because they have an open structure and clear internode position for image composition. Pear shoots stored at 4°C in tissue culture bags were evaluated monthly by standard visual examination and by digital image analysis. Digital images were evaluated for red, green, blue, modified normalized differences of vegetation index (MNDVI), green/red ratio (G/R), intensity, hue, and saturation at the first two nodes of each, plantlet. At 6 mo., the visual ratings had declined steadily for P. communis ‘Luscious’ and ‘Bartlett—Swiss’, while ‘Belle Lucrative’ and ‘Louse Bonne de Jersey’ ratings did not show significant declines until 9 mo. Correlations between visual ratings and G/R and MNDVI values were significant (r2>=0.5) for all cultivars. Regression analysis indicated that the MNDVI and G/R ratios changed significantly over the 15-mo. rating period for most cultivars. Intensity, hue and saturation values were not consistently significant and did not correlate with visual ratings. These results will assist in the development of digital imaging as an alternative technique for evaluation of stored in vitro plantlets.

Culture of CD34+ Umbilical Cord Blood Progenitors with Notch Ligand Results in Enhanced and More Rapid Human Engraftment in a Preclinical NOD/SCID Mouse Model.

Prior clinical studies have indicated that total nucleated and CD34+ cell dose is a critical determinant of hematopoietic reconstitution and survival after unrelated donor umbilical cord blood (UCB) transplantation. Efforts to overcome the problem of small cell numbers in UCB grafts by ex vivo expansion, primarily by culture with cytokines, have not met with success. We have previously shown that culture of CD34+CD38− UCB progenitors with the Notch ligand, Delta1, results in enhanced generation of NOD/SCID repopulating cells. Here we develop a clinically feasible cGMP method for Notch ligand-based expansion of cord blood precursors. Specifically, we investigated the use of CD34+ versus CD34+CD38− cells as a starting population, optimal cytokines and medium, selection of culture vessel and culture period for effects on generation of NOD/SCID repopulating cells. UCB progenitors were cultured in the presence of a Notch ligand form consisting of the extracellular domain of Delta1 fused to the Fc domain of human IgG1 (Delta 1ext-IgG) or control human IgG. Initial studies demonstrated optimal cytokines consisted of SCF, FL, TPO, IL6 and IL3, together with fibronectin fragments in serum free medium. There was no significant difference seen in the CD34 fold expansion with CD34+ versus CD34+CD38− starting cells, however, upon transplantation into NOD/SCID mice, there was a significant increase in the level of human engraftment seen with the CD34+ starting cell population (6.93% vs 2%; p=0.01). Further results from 5 independent experiments in which cord blood CD34+ progenitor cells were cultured for 17 days with immobilized Delta1extIgG or control resulted in a mean fold expansion of CD34+ cells of 230 (± 53) for the Delta cultured cells versus 65 (±31) for the control cultured cells (p=0.03). Delta cultured cells demonstrated significantly enhanced levels of human engraftment as measured by percent CD45 in the marrow of the animals at both 3 weeks (Delta1 15.5%, control 2.6%, uncultured 0.2%; p<0.0001) and at 9 weeks (Delta1 29.4%, control 8.9%, uncultured 7.3%; p<0.0001). We also found significantly greater numbers of SCID repopulating cells (SRC) detected 3 and 9 weeks following transplantation in the Delta1ext-IgG cultured cells compared to control cultured or non-cultured cells (frequency of SRC per 106: 3 weeks, 125 versus 37 or 8, respectively; p=0.0001; 9 weeks, 99 versus 56 or 15, respectively; p=0.01 and p=0.0001). Additional experiments demonstrated that the 17 day culture period was superior to shorter (10 days) or longer (21 days) periods. Relevant to anticipated administration of cultured cord blood units together with a second non-cultured unit in clinical trials, we determined the relative contribution to engraftment of co-infused expanded versus non-manipulated cells in immunodeficient mice, using tissue culture bags as a closed system. We found increased human engraftment in mice that received co-infusions of cultured and uncultured cells compared to either unit alone. Moreover, studies demonstrated that both units contributed to the observed human engraftment suggesting absence of cross-immunologic rejection. This data demonstrate the ability to ex vivo expand UCB repopulating cells using a clinically relevant Notch ligand-based closed culture system and suggests clinical evaluation of this approach to provide more rapid engraftment to overcome the major disadvantage of UCB transplantation.

In Vitro Storage Conditions for Mint Germplasm

Four tissue-cultured mints, Mentha arvensis L., M. spicata L., M. suaveolens Ehrh. hybrid, and M. suaveolens cv. Variegata, were evaluated for survival during storage in media containing three concentrations of N and in four light and temperature regimes. Shoots were placed in plastic, five-chamber, tissue-culture bags on Murashige and Skoog medium (1962) containing 25%, 50%, and 100% of the normal N concentration (MS-N) and stored at 4 °C and –1 °C in darkness, at 4 °C with a 12-hour photoperiod, and at 25 °C with a 16-hour photoperiod. Shoots of all four genotypes stored at 25 °C were in excellent condition after 6 months but required subculture after 18 months. Condition ratings of stored shoots varied with genotype and N concentration. Cultures survived longest at 4 °C with a 12-hour photoperiod on 50% MS-N. Under this regime, all four genotypes were rated in good condition at 30 months but declined to poor condition by 36 months. Based on these data, I recommend that mint cultures be stored on MS medium with 50% MS-N at 4 °C with a 12-hour photoperiod. This regime should provide a minimum of 24 to 36 months of storage before subculture is required. Cold-sensitive genotypes could be stored for 18 months at 25 °C on 50% MS-N medium.

An in Vitro Potato Bioassay to Investigate Tuber Inductive Factors in Continuously Reused Hydroponic Nutrient Solutions

An experiment was conducted in the Biomass Production Chamber (BPC) at Kennedy Space Center to examine the effects of using continuously reused nutrient solution in an NFT system to support potato growth in batch and continuous planting scenarios. Tuberization was hastened and plant growth reduced on plants grown in the aged nutrient solution. We have previously reported that the effect is removed when the aged nutrient solution is filtered through activated charcoal. In order to investigate this apparent plant growth regulator response, an in vitro bioassay has been developed that allows for repeatable, small scale, and rapid testing of the tuber-inducing response. The bioassay is a liquid culture system that employs 600-mL Berzelius beakers capped with modified Sun transparent tissue culture bags, a light shield around the root zone, and a polyurethane foam support, which holds a micropropagated potato plantlet. With this bioassay, we have observed the same plant stunting and tuber initiation effects that were previously seen with the aged nutrient solution. The bioassay appears to be sensitive to environmental factors (PPF, photoperiod, and temperature) that influence tuberization. In addition, partially purified preparations of the apparent growth regulators have elicited the tuberization response. Currently, efforts are underway to examine the role of the microbial community associated with the BPC nutrient delivery system on the tuberization response.

COLD STORAGE OF IN VITRO RUBUS GERMPLASM

In vitro cold storage of Rubus germplasm was investigated using several environmental conditons and types of storage containers. Shoot cultures of Rubus species and cultivars were grown in either tissue culture bags or 20 × 150 mm glass tubes and compared for plant condition and survival under various storage conditions. Cultures stored at 10 C in the dark were in poor condition after 6 months. Cultures kept at 4 C were in much better condition and had higher survival rates after 18 months when stored with a 12 h daylength rather than total darkness. Overall there were no differences in survival or condition between cultures in tubes and bags. Contamination rates were 15% in tubes and 0% in bags. Plants in tissue culture bags could be stored for 9 months at 25 C with 16 h light when the nitrogen level of the MS medium was reduced to 25% and the medium volume was increased from 10 to 20 ml per bag. Genotype differences were apparent under all conditions tested. The best storage condition for Rubus germplasm was 4 C with 12 h light. Plastic tissue culture bags were preferred over tubes due to lower contamination rates.

Generation of Human Lak Cells in Tissue Culture Bags Using Recombinant Il-2 and Serum Free Medium Effects of pretreatment with phenylalanine-methylester

A technique for processing and culturing of human LAK cells using an automated closed system and tissue culture bags is described. To circumvent the inhibitory effects of monocytes on LAK cells the peripheral blood mononuclear cells (PBMC) were pretreated with phenylalanine-methylester (PheOMe). PBMC were obtained from healthy donors by leukapheresis of whole blood. After pretreatment with PheOMe and culturing with IL-2 for 96 h, 60% of the cells remained. PheOMe significantly reduced the number of monocytes (Leu-M3+ cells) from 20-12%. The lytic activity (against K562 and Daudi) of non-PheOMe-treated cells reached a plateau at 72-96 h while PheOMe-treated cells reached maximum activity at 96 h. The total lytic activity/tissue culture bag at 96 h of PheOMe-pretreated cells was significantly augmented in comparison to non-PheOMe-pretreated cells. The present technique allows rapid and simple generation of LAK cells without serum in sterile receptacles suitable for therapy. Additionally, the LAK cell efficacy was improved by reducing the inhibitory effects of monocytes.

Cytotoxic effects in vitro of human monocytes and macrophages on schistosomula of Schistosoma mansoni

Human peripheral blood monocytes from normal donors were isolated by differential centrifugation and cultured in vitro in hydrophobic Teflon-coated tissue culture bags. Cells were harvested between 0 and 10 days and tested for their ability to kill schistosomula of Schistosoma mansoni in an in-vitro cytotoxicity assay. Freshly isolated, unstimulated monocytes demonstrated minimal cytotoxic capability. However, this was increased if the cells were pretreated with human recombinant gamma interferon (IFN-gamma), or with specific anti-S. mansoni antiserum. As the monocytes matured in vitro there were marked increases in the levels of antibody-independent killing of schistosomula. Monocytes grown in vitro with IFN-gamma (10(4) u/ml) took 2-3 days to develop almost maximal cytotoxicity (mean 94% kill of schistosomula). In contrast, unstimulated monocytes (no IFN-gamma) took between 5 and 7 days to achieve comparable cytotoxicity (mean 99% kill). Killing of the schistosomula was dependent upon a high effector to target ratio, and was a relatively slow phenomenon in vitro, parasite attrition occurring between 17 and 36 h. Supernatants from cytotoxic macrophages were ineffective in mediating cytotoxicity of the parasite.