Higher Cell Yields Research Articles

Inline and offline extracorporeal photopheresis: Device performance, cell yields and clinical response.

Extracorporeal photopheresis (ECP) is an effective treatment for graft-vs-host-disease (GvHD). Photopheresis can be performed in offline or inline method. The first uses a conventional cell separator for collection of mononuclear-cells that are photoactivated by a separate device and manually reinfused; the second one involves a dedicated device performing the entire procedure (collection, photoactivation and reinfusion). The objective was to compare the two methods and cell product features to highlight key process, devices performance, and to evaluate ECP clinical response. Patients developing steroid-resistant GvHD underwent ECP as second-line treatment using either inline (Therakos CellEx) or offline system (Terumo BCT Spectra or Optia and UVA PIT system). Data about patients' features, pre-apheresis blood-count, cell product characteristics and clinical response were collected for analysis. We evaluated 494 procedures performed on 28 patients from April 2018 to March 2019. The offline procedure allows to achieve greater cell yield, it is characterized by larger processed blood volume, longer runtime, and higher ACD consumption. The inline procedure shows shorter runtime, high mononuclear-cells percentage and low percentage of granulocytes in cell product. We observed a significant difference in cell yields between inline and offline system; furthermore we did not find a significant relationship between cell dose and clinical response. Inline ECP is fast, highly automated and productive, making it particularly suitable for ECP treatments. Offline ECP collects high cell yields implying longer procedure and greater operator intervention. Our study did not find a significant relationship between cell dose and GVHD response.

Dystrophin Dp71ab is monoclonally expressed in human satellite cells and enhances proliferation of myoblast cells

Dystrophin Dp71 is the smallest isoform of the DMD gene, mutations in which cause Duchenne muscular dystrophy (DMD). Dp71 has also been shown to have roles in various cellular processes. Stem cell-based therapy may be effective in treating DMD, but the inability to generate a sufficient number of stem cells remains a significant obstacle. Although Dp71 is comprised of many variants, Dp71 in satellite cells has not yet been studied. Here, the full-length Dp71 consisting of 18 exons from exons G1 to 79 was amplified by reverse transcription-PCR from total RNA of human satellite cells. The amplified product showed deletion of both exons 71 and 78 in all sequenced clones, indicating monoclonal expression of Dp71ab. Western blotting of the satellite cell lysate showed a band corresponding to over-expressed Dp71ab. Transfection of a plasmid expressing Dp71ab into human myoblasts significantly enhanced cell proliferation when compared to the cells transfected with the mock plasmid. However, transfection of the Dp71 expression plasmid encoding all 18 exons did not enhance myoblast proliferation. These findings indicated that Dp71ab, but not Dp71, is a molecular enhancer of myoblast proliferation and that transfection with Dp71ab may generate a high yield of stem cells for DMD treatment.

Microcarrier expansion of c-MycERTAM -modified human olfactory mucosa cells for neural regeneration.

Human olfactory mucosa cells (hOMCs) have potential as a regenerative therapy for spinal cord injury. In our earlier work, we derived PA5 cells, a polyclonal population that retains functional attributes of primary human OMCs. Microcarrier suspension culture is an alternative to planar two-dimensinal culture to produce cells in quantities that can meet the needs of clinical development. This study aimed to screen the effects of 10 microcarriers on PA5 hOMCs yield and phenotype. Studies performed in well plates led to a 2.9-fold higher cell yield on plastic compared to plastic plus microcarriers with upregulation of neural markers β-III tubulin and nestin for both conditions. Microcarrier suspension culture resulted in concentrations of 1.4 × 105 cells/ml and 4.9 × 104 cells/ml for plastic and plastic plus, respectively, after 7 days. p75NTR transcript was significantly upregulated for PA5 hOMCs grown on Plastic Plus compared to Plastic. Furthermore, coculture of PA5 hOMCs grown on Plastic Plus with a neuronal cell line (NG108-15) led to increased neurite outgrowth. This study shows successful expansion of PA5 cells using suspension culture on microcarriers, and it reveals competing effects of microcarriers on cell expansion versus functional attributes, showing that designing scalable bioprocesses should not only be driven by cell yields.

In vitro megakaryocyte culture from human bone marrow aspirates as a research and diagnostic tool

Megakaryocytes (MKs) are relatively rare in bone marrow, comprising <0.05% of the nucleated cells, which makes direct isolation from human bone marrow impractical. As such, in vitro expansion of primary MKs from patient samples offers exciting fundamental and clinical opportunities. As most of the developed ex vivo methods require a substantial volume of biomaterial, they are not widely performed on young patients. Here we propose a simple, robust, and adapted method of primary human MK culture from 1 mL of bone marrow aspirate. Our technique uses a small volume of bone marrow per culture, uses straightforward isolation methods, and generates approximately 6 × 105 mature MKs per culture. The relative high cell purity and yield achieved by this technique, combined with efficient use of low volumes of bone marrow, make this approach suitable for diagnostic and basic research of human megakaryopoiesis.

Intracoronary injection of haematopoietic precursor cells regenerates the borders, but not the core, of old myocardial scars.

AimsCell therapy regenerative potential is hindered by cell access to the infarct zone. We studied function recovery at the scar zone and its impact in global left ventricular function after intracoronary injection of haematopoietic precursor cells.Methods and resultsHaematopoietic precursor cells were obtained by blood apheresis in patients with an old myocardial infarction, and the presence of CD34+ and CD133+ cells was quantified. Left ventricular function, volumes, and infarct zone segmental motion were measured by magnetic resonance imaging (MRI) and echo left ventricular segmental strain (LVSS). The aphaeresis product was administered to 20 patients in the coronary artery responsible for the myocardial infarction. High cell yield in blood aphaeresis product allowed us to inject a high number of cells in most patients. Three patients were excluded because of insufficient CD133+ cell number, and one more patient was excluded because of artefacts in MRI images. The remaining 16 patients were compared with 16 controls. After 1 year, infarct zone reduction was related to the number of CD133+ (R = 0.53; P %3C 0.05) and CD34+ (R = 0.63; P %3C 0.01) cells injected. The number of CD133+ cells injected was also related to an improvement in LVSS (R = 0.62; P %3C 0.01). In turn, scar zone reduction was related to an improvement in LVSS (R = 0.64; P %3C 0.01). End‐diastolic volume showed a reduction at follow‐up in the treated group when compared with control patients. MRI infarct area segments systolic thickness increase improved after treatment in treated patients [expressed as median (interquartile range)] [0.42 (−0.38 to 1.14) vs. 1.06 (−0.10 to 2.12) mm; P %3C 0.01], but not in controls [2.02 (0.75 to 3.4) vs. 1.91 (0.77–3.17) mm; P = not significant (n.s.)]. In cell therapy patients, the borders of the infarct zone, but not the core, showed a significant recovery [proximal rim: 0.48 (−0.18 to 1.33) vs. 1.07 (0.22–2.40) mm; P %3C 0.05, distal rim: 0.75 (0.26–1.40) vs. 1.76 (0.65–2.86) mm; P %3C 0.05, and core: 0.36 (−0.33 to 1.20) vs. 0.60 (−0.18 to 1.62) mm; P = n.s.]. That improvement was not observed in the control group [proximal rim: 1.20 (0.33–2.53) vs. 0.82 (−0.13 to 1.65) mm; P = n.s., distal rim: 1.24 (0.80–1.72) vs. 0.96 (0.19–1.81) mm; P = n.s., and core: 0.30 (−0.42 to 1.64) vs. 0.07 (−0.60 to 1.20) mm; P = n.s.]. Only small size infarcts showed a complete recovery in the cell therapy patients [systolic thickness increase post‐treatment increment in infarcts ≤6 segments vs. >6 segments affected: 0.28 (−0.19 to 0.71) vs. −1.21 (−2.60 to −0.53) mm; P %3C 0.01].ConclusionsIntracoronary injection of peripheral blood‐derived haematopoietic precursor cells produces a complete recovery of the borders and partial regeneration of the infarct core, which is directly related to the number of CD133+ and CD34+ cells injected. Cell therapy infarct zone regeneration prevents ventricular remodelling by preserving segmental contractility and halting left ventricular dilatation.

Comparison and optimization of extraction methods of extracellular polymeric substances in anammox granules: From maintaining protein secondary structure perspective

Extracellular proteins are crucial for the formation and structural maintenance of anammox granules. However, the influence of extracellular polymeric substances (EPS) extraction methods on the secondary structure of extracellular proteins has not been studied fully. In this work, ten EPS extraction methods were applied to anammox granules and compared systematically. The EPS extraction methods were then applied to bovine serum albumin (BSA), a representative of extracellular proteins, to investigate their influence on protein secondary structure by circular dichroism (CD) spectroscopy. The ten EPS extraction methods yielded EPS concentration varying from 5.2 ± 0.1 to 84.8 ± 2.0 mg/g volatile suspended solid (VSS). Tween-20 treatment resulted in the most similar CD spectrum and protein secondary structure to the control. Treatment 0.5% Tween-20 for 4 h was found to be the best condition to preserve protein secondary structure while achieving a high EPS yield (44.4 ± 1.4 mg/g VSS) and limited cell lysis for anammox granules. This study provides an optimized protocol to extract granular EPS when the role of the secondary structure of extracellular proteins in regulating aggregation, adsorption and biofilm formation of microbes is to be elucidated.

High yield of human umbilical cord-derived mesenchymal stromal cells in an animal component-free culture medium

Background & Aim Mesenchymal stromal cells (MSCs) can be isolated from adult and new born tissue sources. Unlike adult bone marrow, the collection of human umbilical cord (UC) tissue offers the advantage of being noninvasive. MSCs isolated from UC tissues are also not negatively impacted by advanced age and certain diseases, which serve their banking for future clinical applications. We have optimized MesenCult™-ACF Plus (MACFP), an improved animal component-free (ACF) culture medium for the efficient derivation and expansion of MSCs from UC tissue. Methods, Results & Conclusion Wharton's Jelly was dissected from the UC, cut into 1-2 mm3 fragments and placed into individual wells of a 24-well pre-coated tissue culture plate in UC-MesenCult™-ACF Plus (UC-MACFP) medium supplemented with an optimized cytokine cocktail, or in control medium containing fetal bovine serum (FBS). MSCs migrated from the explanted fragments in 10-14 days and were replated at 1.5–3.0 × 103 cells/cm2 for long-term expansion in each medium. The proliferative potential of UC-derived MSCs was measured by counting cells at each passage (P) up to P6 in both media and their phenotype was assessed. MSC differentiation potential was examined by culturing these cells in adipogenic and osteogenic differentiation media. The average yield of MSCs from UC was higher in UC-MACFP than in FBS medium (9.9±3.4 × 105 vs. 4.4±1.8 × 105 cells/g [mean ± SEM]; n=11 and 5, respectively). Cell outgrowth was further increased to 2.6±1.0 × 106 cells/g (n=9) with addition of 2.5% human AB serum to UC-MACFP medium. The average fold-expansion of UC-MSCs at each subculture over 6 passages was comparable in UC-MACFP (13.5 ± 0.9; n=13) and in FBS media (12.8±0.6; n=6). The phenotype of MSCs was similar in both UC-MACFP and FBS media, with >95% of cells positively expressing CD105, CD73 and CD90, and

MAPC® cell therapy enhances the ex-vivo expansion of polyclonal, regulatory T cells

Background & Aim Regulatory T (Treg) cells are a naturally occurring population of lymphocytes that maintain immune homeostasis and resolve excessive immune activation. They act through a spectrum of cell-mediated and soluble mechanisms. Preclinical data in humanized mouse models has firmly established that adoptive transfer or in vivo generation of Treg cells can remedy autoimmunity, GVHD and solid organ transplant rejection; a few clinical trials have already demonstrated safety and suggest some limited efficacy. Therefore, clinical adoptive Treg cell therapy (the isolation, ex vivo expansion and re-infusion of Treg cells) has been heralded as a promising strategy for the treatment of auto-immune and inflammatory disorders. As such, a living drug cell therapy may have greater specificity and more complex therapeutic benefits than conventional immunosuppressive drugs and could potentially cure autoimmune diseases by restoring the immune tolerance. However, several major roadblocks in the widespread clinical implementation of Treg therapy remain, mainly the low number of Treg cells that can be harvested from peripheral blood (PB) and the limited expansion potential, while maintaining purity and function. Methods, Results & Conclusion ReGenesys has developed a platform technology for the expansion of Treg cells derived from PB while fully maintaining their functional characteristics. This platform is based on the use of our proprietary MAPC stromal stem cell therapy which consists of non-hematopoietic stem cells derived from bone marrow that secrete various growth factors and have potent immunomodulatory effects. When CD4+CD25+127low-sorted Treg cells from PB were expanded on MAPC cells, a robust and high Treg cell yield was obtained, reaching the critical threshold of clinical batch production (1 billion cells) in all donors. Furthermore, a higher purity was observed (less potential CD8+ T cell contamination) and a specific signature was seen in terms of phenotype (low skin-homing/ high gut-homing marker expression). The immunosuppressive function was confirmed in both in vitro and in vivo models. Finally, successful Treg expansions have also been performed from PB of Crohn's Disease patients.

Desirability profiling to rank scalable mesenchymal stromal cell enhancement methods according to multimodal mechanisms

Background & Aim Mesenchymal stromal cells (MSCs) are widely investigated due to their anti-inflammatory, angiogenic, and anti-fibrotic properties. However, insufficient potency, MSC donor variability, and deficient knowledge on therapeutic mechanisms can limit MSC treatment efficacy. The Viswanathan lab has pioneered a novel, non-genetic proprietary strategy to culture MSCs in 3D aggregates under serum-free conditions to augment anti-inflammatory functions. Hypoxic culture has also been shown to enhance MSC paracrine functions. Our objective is to systematically characterize the anti-inflammatory, angiogenic, and anti-fibrotic properties of 3D and hypoxic MSCs, and to apply desirability profiling to empirically rank the potency of the MSCs according to these different properties. Methods, Results & Conclusion METHODS: Human adipose tissue (AT) or bone marrow (BM) MSCs were expanded with serum-free medium. MSCs were then cultured for 24-h under 3D (static culture or in 0.1 L vertical-wheel bioreactors) or 2D hypoxic (5% O2) conditions, with 2D normoxic MSCs as controls. Gene expression was measured by RT-qPCR and using a NanoString 50-gene panel, while microRNAs (miR) were examined by miR-sequencing. In ongoing work, desirability profiling will be performed on these data and on additional functional read-outs in MSC co-cultures with human monocytes, endothelial cells, or fibroblasts. RESULTS 3D MSC culture under static and bioreactor conditions resulted in similar high viable cell yields (>80%). 3D MSCs had increased expression of immunomodulatory genes (e.g. IDO, TSG6 with 4- to 10-fold increased expression vs. 2D normoxic control) while hypoxic MSCs had greater expression of angiogenic genes (e.g. VEGF with 2-fold greater expression vs. control). Preliminary investigation into miR expression revealed that AT-MSCs displayed a distinct miR profile relative to BM-MSCs, and that 3D enhanced AT-MSCs had a differential miR profile vs. controls. The miRs and their target pathways are being investigated to identify mechanisms underlying the enhanced MSC functionality. CONCLUSION MSCs act through multimodal mechanisms that are enhanced through 3D or hypoxic cultures. Statistical modeling is being used to systematically rank the potency attributes of enhanced MSCs in terms of their anti-inflammatory, angiogenic, and anti-fibrotic functionality in vitro and in inflammatory mouse models. Ultimately, this work will generate methods for improved MSC products with therapeutic profiles that are “fit-for-purpose.”

Feasibility of defined, animal component free, protein free, salt base freezing solution composed of 5% DMSO for cryopreservation of HMSC toward clinical applications

Background & Aim hMSC are a critical raw material for cell-based therapies and usually a large number of cells are required for clinical applications. Cryopreservation is currently the only method to preserve cells with maintained functional properties and genetic stability for long term storage. To date, a common practice is use of homebrew cryo-medium that is composed of animal-derived raw materials and low Mw Cryoprotective agents (CPAs), usually 10-20% DMSO. Alternatively, animal component free (ACF) freezing solutions were developed for clinical applications, but they are still based on culture medium and are composed of 10% DMSO (e.g. NutriFreez™ D10). In addition, a few products were developed with a reduced concentration of DMSO or even without DMSO. However, these products appear not to be optimal for hMSC. Moreover, most of the commercially available DMSO-free products are actually composed of other potentially toxic permeable CPAs (e.g. Ethylene Glycol, EG). Exposure of cells to these materials can impact the quality, safety and efficacy of cell-based therapeutic product. Facing strict regulatory requirements, the development of defined, ACF, salt base freezing solution with reduced concentration of DMSO is essential. Methods, Results & Conclusion The current study presents the feasibility of a novel defined, ACF, protein free, salt base freezing solution composed of 5% DMSO for cryopreservation of hMSC from various sources. Results show that the novel cryopreservation solution efficiently maintains high cell yield and viability, and supports the recovery of hMSC while maintaining the normal hMSC features: typical fibroblast-like cell morphology, phenotypic surface marker profile, differentiation capability as well as self-renewal potential. Clinically accepted, cryopreservation solution for hMSC holds a unique opportunity to facilitate the translation of these cells to cellular therapy applications.

CD146+CD107ahigh define a highly secretory and immunosuppressive Mesenchymal Stem/Stromal Cells subset with enhanced therapeutic effects reversing synovitis and fat pad fibrosis through M1/M2 phenotypic shift

Background & Aim Mesenchymal stem/stromal cells (MSC) are highly immunomodulatory and promising therapeutic candidates for numerous clinical indications. Translational advances of cell-based therapies require standardization, thus intrinsic heterogeneity of MSC may warrant concerns and yield inconsistent outcomes. Human bone marrow-derived MSC (BM-MSC) subpopulations have been discriminated based on their specific niche, however thorough functional analyses correlating potencies has yet to be determined. Methods, Results & Conclusion Methods Herein, we investigated the comprehensive functionality of BM-MSC (n=8) discerned by perivascular marker CD146, inadvertently average in crude BM-MSC outcomes. Initially, inflammatory challenge (i.e., priming) to crude BM-MSC, termed unfractionated (UNF), captured a baseline of signature responses including enhanced CD146+ expression. So, UNF preparation and immunoselected subpopulations based on CD146 positivity (CD146+ & CD146− cohorts) were assessed for phenotypic, transcriptional and secretome profiles, and in vitro/in vivo functional evaluations (immunopotency assay and reversing inflammatory and fibrotic events induced in the rat knee). Results Both primed UNF and CD146+ sorted cells enriched for a CD107ahigh, CXCR4high, and LepRhigh phenotype.Molecular evaluations evidenced CD146+ cells (not the CD146− cohort) exhibiting a highly immunomodulatory transcriptome and secretome, with enhanced secretory capacity (determined by CD107aHigh). This was functionally evident in robust immunosuppression of both stimulated PBMC and T lymphocytes while inducing significant frequencies of Regulatory T cells, providing evidence of CD146+CD107ahigh BM-MSC as the distinctively potent responders to injury and inflammation. This was supported in vivo using a rat model of knee synovium and infrapatellar fat pad (IFP) inflammation and fibrosis, efficiently reversed following CD146+ BM-MSC treatment (even at reduced cell doses – 1/10th) and largely failed by the CD146− counterpart. Mechanistically, CD146+ cells greatly promoted a pivotal M1-to-M2 macrophage phenotypic shift within synovium and IFP further supporting local immunomodulatory effects. Conclusion This study definitively supports that within crude BM-MSC, the CD146+CD107ahigh subpopulation, deemed ‘first responders’ facilitate high therapeutic efficacy. By selection of the CD146+ BM-MSC, our evidence implicates a highly translatable method that may reduce the need for high cell yields while improving outcomes.

Long‐term cultivation of human amniotic fluid stem cells: The impact on proliferative capacity and differentiation potential

Human amniotic fluid mesenchymal stem cells (AF-MSCs) are a valuable, easily obtainable alternative source of SCs for regenerative medicine. Usually, amounts of cells required for the translational purposes are large thus the goal of this study was to assess the potency of AF-MSCs to proliferate and differentiate during long-term cultivation in vitro. AF-MSCs were isolated from amniotic fluid of healthy women in the second trimester of pregnancy and cultivated in vitro. AF-MSCs were cultivated up to 42 passages and they still maintained pluripotency genes, such as OCT4, SOX2, and NANOG, expression at a similar level as in the initial passages as determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Fluorescence-activated cell sorting analysis demonstrated that the cell surface markers CD34 (negative), CD44, and CD105 (positive) expression was also stable, only the expression of SCs marker CD90 decreased during the cultivation. The morphology of AF-MSCs changed over passage, acridine orange/ethidium bromide staining revealed that more cells entered into apoptosis and the first signs of aging were detected only at late passages (later than p33) using SA-β-gal assay. Concomitantly, the differentiation potential towards cardiomyogenic lineage, induced with DNA methyltransferases inhibitors decitabine, zebularine, and RG108, was impaired when comparing AF-MSCs at p31/33 with p6. The expression of cardiomyocytes genes MYH6, TNNT2, DES together with ion channels genes of the heart (sodium, calcium, and potassium) decreased in p31/33 induced AF-MSCs. AF-MSCs have a great proliferative capacity and maintain most of the characteristics up to 33 passages; however, the cardiomyogenic differentiation capacity decreases to a certain extent during the long-term cultivation. These results provide useful insights for the potential use of AF-MSCs for biobanking and broad applications requiring high yield of cells or repeated infusions. Hence, it is vital to take into account the passage number of AF-MSCs, cultivated in culture, when utilizing them in vivo or in clinical experiments.

The Preparation of Chicken Kidney Cell Cultures for Virus Propagation.

Chicken kidney (CK) cells have been widely utilized in virus research studies for many years. The optimized technique of primary CK cell culture production involving both mechanical and enzymatic disaggregation is described. This updated method proved to consistently give high cell yields and resultant cultures are readily used for virus assays.

A simple method for in vitro preparation of natural killer cells from cord blood

BackgroundCord Blood (CB) has been considered a promising source of natural killer (NK) cells for cellular immunotherapy. However, it is difficult to expand the large numbers of highly pure NK cells from CB without cell sorting and feeder cells/multiple cytokines. In this study, we try to develop a simple, safe and economical method for ex vivo expansion and purification of NK cells from CB without cell sorting and feeder cells/multiple cytokines.ResultsThe large numbers (mean: 1.59 × 1010) of highly pure (≥90%) NK cells from CB could be obtained through interleukin-2, group A streptococcus and zoledronate stimulation of mononuclear cells using the 21-day culture approach. When compared to resting NK cells, expanded NK cells were a higher expression of activating receptors CD16, NKG2D, NKp30, NKp44, NKp46 and activating markers CD62L and CD69, while the inhibitory receptors, CD158a and CD158b remained largely unchanged. In addition, these cells showed a higher concentration of IFN-γ, TNF-α and GM-CSF secretion and cytotoxicity to K562 cells and acute myeloid leukemia targets than resting NK cells.ConclusionWe develop a simple, safe and economical method to obtain high yield, purity, and functionality NK cells from CB without cell sorting and feeder cells/multiple cytokines.

Towards a Standardization and Characterization of Clinical Grade Adipose-Derived Stromal Cells

INTRODUCTION Mesenchymal Stromal Cells (MSCs) are multipotent cells with regenerative, anti-inflammatory, immunomodulatory and anti-tumorigenic properties, which are readily isolated from aspirates of adipose tissue (AT) due to their high availability. Compared with alternative sources, AT provides high cell yield. Despite the numerous clinical trials in autologous and allogeneic settings for various diseases worldwide, there are no US FDA-approved MSC-based products. Challenges for clinical translation of MSC-based therapies largely lie in the sourcing, production and basic characterization of such products (Camilleri et al, 2016; Mendicino et al, 2014). Given the known donor-related variability (sex, age, liposuction sites, BMI, etc.), there is still ample room to: better standardize in vitro manufacturing processesrefine the characterization of clinical products. METHODS Seven samples of lipoaspirate were processed. AT manual digestion was compared with an automated collagenase-based enzymatic digestion by the Cytori Celution instrument, a closed system regulated as a medical device by EC for the production of adipose-derived stem cells-enriched products. Total cell yield, cell harvest at P0, P1 and P2, Population Doubling Level (PDL) and Doubling Time (DT) were evaluated. The obtained Adipose-derived Stromal Cells (ADSCs) were characterized for: Expression of 361 surface markers (BioLegend LegendScreen Human PE Kit), including 117 additional antigens compared to other reported screens. Cytori-isolated ADSCs from three donors for 6 total conditions (3 P2, 2 P3 and 1 IFN-g-primed P2) were screened. A broader characterization of fresh isolates (P0) as well as (P3) from the same donors was performed with a 20-color flow cytometric panel. Data were analyzed with FlowJo software.miRNA expression was evaluated using the 800 human miRNAs Nanostring panel. Nanostring nCounter Analysis System generated data, then Partek GS software was used for secondary analysis. RESULTS Cytori processing resulted in a higher cell yield at isolation (p= 0.0156), and ADSCs exhibited higher proliferation in vitro at P0 (n. of days at P0, p=0.0313), higher cell yield at P1 (p=0.0313), and higher PDL1 (p=0.0469). Out of the 361 markers examined, in every donor at each passage, at least 94 were found positive (expression &gt;1%), including 30 within the newly screened 117 markers. Altogether, 27 markers were expressed at more than 85%, including 6 from the newly screened markers. Our analysis identified 50 donor-dependent markers, 50 passage-modulated markers (P2 vs P3) and 45 IFN-g-modulated marker, 41 increased and 4 decreased. We were able to identify the ADSCs progenitors markers CD146 and CD271 on cell as in P0 and P3, we observed a transition from a dominant expression in P0 of CD271 (34.05%) vs CD146 (1.62% ) to a dominant expression of CD146 in P3 (52%) vs CD271 (0.7%) in a single donor. The frequency of double positives CD146+CD271+ was 1% and 0.6% in P0 and P3 respectively. miRNA expression analysis by ANOVA revealed that (i) IFN-g-priming modulated 19 miRNAs, that 4 miRNAs were modulated by passage number and donors' origin. CONCLUSION Our study shows that the standardized Cytori processing advantageously substitutes AT manual digestion by enabling higher cell yield and potentially providing multiple ADSC doses from a single donor. The isolation procedure is standardized, the operator-dependent variations are minimized, and less prone to contamination compared to the lengthy, multistep process of manual digestion. The broad cell characterization with flow cytometry and miRNA expression revealed the expression and modulation of new markers. We believe that increasing the dimensionality of the ADSC characterization, beyond the traditional markers, could reveal markers that describe specific functional abilities of clinical ADSC product. Proper functional studies are necessary to validate our hypothesis. Disclosures No relevant conflicts of interest to declare.

Optimization of a suspension culture for a Theileria annulata-infected bovine cell line

Theileria annulata schizont transformed bovine lymphocytes show the feature of permanent proliferation in vitro culture. In this study, we optimized a suitable culture medium for transformed cells to ensure a high yield of quality cells in suspension culture. As the basis for the optimized medium, we combined 75% Gibco (GB) and 25% RPMI-1640 medium. Glucose, lactic acid, ammonia, growth factors and several kinds of amino acids at specific concentrations play important roles in maintaining the maximum growth rate and the quality of cells. The metabolic flow of 17 amino acids, glucose and nutrients was determined with high-performance liquid chromatography (HPLC) and cell viability analysis. The genetic stability of the TaSP and TaSE genes at different passages of the cell line in suspension culture was determined using PCR amplification. The optimal concentrations or tolerated levels of glucose, lactic acid and ammonia were 10–14, 2–5.5 and 3.5–5.5 mmol/L, respectively. Our data demonstrated that the potential utility of the medium optimized here to yield high quality cells compared with basal (normally used) medium. The medium also facilitated the easy maintenance of transformed cells with high yields and excellent quality for in vitro studies. This study also provides insight into the processes of optimization and vaccine development.

Overproduction and purification of highly active recombinant Pseudomonas aeruginosa str. PAO1 RNA polymerase holoenzyme complex

The bacterial RNA polymerase (RNAP) is a large, complex molecular machine that is the engine of gene expression. Despite global conservation in their structures and function, RNAPs from different bacteria can have unique features in promoter and transcription factor recognition. Therefore, availability of purified RNAP from different bacteria is key to understanding these species-specific aspects and will be valuable for antibiotic drug discovery. Pseudomonas aeruginosa is one of the leading causes of hospital and community acquired infections worldwide - making the organism an important public health pathogen. We developed a method for producing high quantities of highly pure and active recombinant P. aeruginosa str. PAO1 RNAP core and holoenzyme complexes that employed two-vector systems for expressing the core enzyme (α, β, β’, and ω subunits) and for expressing the holoenzyme complex (core + σ70). Unlike other RNAP expression approaches, we used a low temperature autoinduction system in E. coli with T7 promoters that produced high cell yields and stable protein expression. The purification strategy comprised of four chromatographic separation steps (metal chelate, heparin, and ion-exchange) with yields of up to 11 mg per 500 mL culture. Purified holoenzyme and reconstituted holoenzyme from core and σ70 were highly active at transcribing both small and large-sized DNA templates, with a determined elongation rate of ~18 nt/s for the holoenzyme. The successful purification of the P. aeruginosa RNAP provides a gateway for studies focusing on in vitro transcriptional regulation in this pathogen.

High cell density cultivation of Lipomyces starkeyi for achieving highly efficient lipid production from sugar under low C/N ratio

Oleaginous microorganisms such as oleaginous yeasts are well known for the ability to accumulate intracelular lipid more than 20% w/w in certain conditions. Lipomyces starkeyi D35 (Ls-D35 strain) is one of promising oleaginous yeast for lipid production. The purpose of this research is to establish the optimum condition for cell and lipid production of Ls-D35 strain under low C/N ratio (17.9) by high cell density cultivation. The optimum pH for cell and lipid production was achieved at pH 5 by using batch cultivation. Four different feeding strategies were conducted to observe the cell and lipid production of Ls-D35 strain. The highest cell yield was achieved by mixed glucose and xylose as substrates (0.15 w/w substrate) after 96 h and the highest lipid yield was achieved by single xylose feeding (0.13 w/w substrate) after 120 h. Meanwhile, the two most fatty acids content were C18:1 and C16:0 which are similar to the content of vegetable oil. These results showed that Ls-D35 strain could produce high cell and optimum lipid under low initial C/N ratio medium which can be an alternative for biodiesel production and other industrial products.

Isolation and expansion of high yield of pure mesenchymal stromal cells from fresh and cryopreserved placental tissues

The injection of placental stromal cells isolated from fetal human tissues (f-hPSC) was reported to indirectly induce tissue regeneration in different animal models. A procedure of f-hPSC isolation from fragments of both selected fresh or cryopreserved bulk placental neonate tissues is proposed, based on their high migratory potential,. The fragments of the desired fetal placental tissues are adhered to a culture dish by traces of diluted fibrin and covered with culture medium. Spontaneous migration of pure f-hPSC from the tissue fragments to the cell culture dishes is followed by their rapid expansion by numerous passages. The isolated f-hPSC express typical mesenchymal surface antigens, including CD29, CD105, CD166 and CD146, with negative expression of white blood cell lineage and endothelial cells markers. Optimal yields of f-hPSC cultures can also be obtained from tissue samples cryopreserved in medium composed of 10% dimethyl sulfoxide (M2SO) and 50% fetal calf serum. Slightly better yields are obtained with media supplemented with 1% human albumin. Medium with 5% M2SO and/or 0.25 mg/ml PEG yielded inferior results. The f-hPSC from fresh or cryopreserved tissues express similar cell markers and growth kinetics. The proposed isolation protocol may also be applied for high yield isolation of stromal cells from fresh and cryopreserved tissue of other organs.

Tide motion bioreactors for large-scale cultivation and expansion of human mesenchymal stem cells

Background & Aim The future cell therapy demands high quantities of MSCs ranging from 10 million to more than 200 million cells per dosage. Conventional expansion of MSCs on plasticwares become impractical when large dosages of more than 50 million cells are required. Commercial manufacturing for large quantities of cells at a high quality and acceptable cost becomes critical. The use of bioreactors culture which combines process control, up-scalability and automation is the primary solution for this need. Small scale reactors are often used in the early developmental stages of bioconversion processes. Many MSCs bioreactor-based manufacturing processes are complicated by the inability to separate cells from micro/macro carriers with high cell yield and viability. Functional and phenotypic characteristics are lost during harvesting of cells from microcarriers. Here we aim to optimize key parameters such as cell seeding densities, media culturing conditions, harvesting procedures and various bioprocesses commonly examined in bioreactors. Methods, Results & Conclusion Esco Aster has leveraged on the Tide Motion bioreactors to establish a robust and scalable platform using macrocarriers to meet the demands for future clinical therapies. MSCs isolated from different lineages were seeded and allowed to expand within PET macrocarriers while cell counts were measured and monitored daily. Extracellular matrices secreted by MSCs was observed in culture, which promote more physiological-like conditions and aid in the expansion of MSCs and maintaining their physiological characteristics. Throughout culture periods, cell culture conditions were monitored, with bioprocess parameters such as glucose consumption and pH levels measured to ensure proper scale-up. Key issues such as scaffold coatings, cell seeding densities, media culturing conditions, and improved bioprocess parameters needed for optimal stem cell systems were studied in our system. Overall, we present our process optimization with quality controls and release criteria of functional and phenotypic characteristics for the translation of academic/industrial R&D into bench sale for future clinical trials and commercialization process.