Ideal Culture Conditions Research Articles

Optimizing recombinant antibody fragment production: A comparison of artificial intelligence and statistical modeling.

Maximizing the recombinant protein yield necessitates optimizing the production medium. This can be done using a variety of methods, including the conventional "one-factor-at-a-time" approach and more recent statistical and mathematical methods such as artificial neural network (ANN), genetic algorithm, etc. Every approach has advantages and disadvantages of its own, yet even when a technique has flaws, it is nevertheless used to get the best results. Here, one categorical variable and four numerical parameters, including post-induction time, inducer concentration, post-induction temperature, and pre-induction cell density, were optimized using the 232 experimental assays of the central composite design. The direct and indirect effects of factors on the yield of anti-epithelial cell adhesion molecule extracellular domain fragment antibody were examined using statistical methods. The analysis of variance results indicate that the response surface methodology (RSM) model is effective in predicting the amount of produced single-chain fragment variable (p-value = 0.0001 and R2 = 0.905). For ANN modeling, the evaluation using normalized root mean square error (NRMSE) and R2 values shows a good fit (R2 = 0.942) and accurate predictions (NRMSE = 0.145). The analysis of error parameters and R2 of a dataset, which contained 30 data points randomly selected from the complete dataset, showed that the ANN model had a higher R2 value (0.968) compared to the RSM model (0.932). Furthermore, the ANN model demonstrated stronger predictive ability with a lower NRMSE (0.048vs. 0.064). Induction at the cell density of 0.7 and an isopropyl β-D-1-thiogalactopyranoside concentration of 0.6mM for 32 h at 30°C in BW25113 was the ideal culture condition leading to the protein yield of 259.51mg/L. Under the optimum conditions, the output values predicted by the ANN model (259.83mg/L) were more in line with the experimental data (259.51mg/L) than the RSM (276.13mg/L) expected value. This outcome demonstrated that the ANN model outperforms the RSM in terms of prediction accuracy.

Defining the Most Potent Osteoinductive Culture Conditions for MC3T3-E1 Cells Reveals No Implication of Oxidative Stress or Energy Metabolism.

The MC3T3-E1 preosteoblastic cell line is widely utilised as a reliable in vitro system to assess bone formation. However, the experimental growth conditions for these cells hugely diverge, and, particularly, the osteogenic medium (OSM)'s composition varies in research studies. Therefore, we aimed to define the ideal culture conditions for MC3T3-E1 subclone 4 cells with regard to their mineralization capacity and explore if oxidative stress or the cellular metabolism processes are implicated. Cells were treated with nine different combinations of long-lasting ascorbate (Asc) and β-glycerophosphate (βGP), and osteogenesis/calcification was evaluated at three different time-points by qPCR, Western blotting, and bone nodule staining. Key molecules of the oxidative and metabolic pathways were also assessed. It was found that sufficient mineral deposition was achieved only in the 150 μg.mL-1/2 mM Asc/βGP combination on day 21 in OSM, and this was supported by Runx2, Alpl, Bglap, and Col1a1 expression level increases. NOX2 and SOD2 as well as PGC1α and Tfam were also monitored as indicators of redox and metabolic processes, respectively, where no differences were observed. Elevation in OCN protein levels and ALP activity showed that mineralisation comes as a result of these differences. This work defines the most appropriate culture conditions for MC3T3-E1 cells and could be used by other research laboratories in this field.

A matter of differentiation: equine enteroids as a model for the in vivo intestinal epithelium

Epithelial damage due to gastrointestinal disorders frequently causes severe disease in horses. To study the underlying pathophysiological processes, we aimed to establish equine jejunum and colon enteroids (eqJE, eqCE) mimicking the in vivo epithelium. Therefore, enteroids were cultivated in four different media for differentiation and subsequently characterized histomorphologically, on mRNA and on protein level in comparison to the native epithelium of the same donor horses to identify ideal culture conditions for an in vitro model system. With increasing enterocyte differentiation, the enteroids showed a reduced growth rate as well as a predominantly spherical morphology and less budding compared to enteroids in proliferation medium. Combined or individual withdrawal of stem cell niche pathway components resulted in lower mRNA expression levels of stem cell markers and concomitant differentiation of enterocytes, goblet cells and enteroendocrine cells. For eqCE, withdrawal of Wnt alone was sufficient for the generation of differentiated enterocytes with a close resemblance to the in vivo epithelium. Combined removal of Wnt, R-spondin and Noggin and the addition of DAPT stimulated differentiation of eqJE at a similar level as the in vivo epithelium, particularly with regard to enterocytes. In summary, we successfully defined a medium composition that promotes the formation of eqJE and eqCE consisting of multiple cell types and resembling the in vivo epithelium. Our findings emphasize the importance of adapting culture conditions to the respective species and the intestinal segment. This in vitro model will be used to investigate the pathological mechanisms underlying equine gastrointestinal disorders in future studies.

Response Surface Methodology Approach to Optimize the Expression of Thioredoxin-MOG Fusion Protein

Background: The N-terminal domain of the myelin oligodendrocyte glycoprotein (MOG) has been shown to generate experimental autoimmune encephalomyelitis (EAE), an animal model of MS. A considerable amount of MOG must be accessible for EAE induction. Here, for the first time, Response Surface Methodology-Box-Behnken (RSM-BBD) was employed to identify the ideal culture conditions for causing Escherichia coli (E. coli) BL21 to overproduce the Thioredoxin-MOG (Trx-MOG) fusion protein. The RSM method is a powerful, efficient, and reliable alternative to the One-Factor-At-A-Time (OFAT) method in optimizing process variables, allowing for a smaller number of experimental runs, investigating variable interaction, and being cheaper and less time-consuming. Methods: Here, using the 29 experimental assays, the direct and indirect effects of factors including post-induction time, IPTGinducer concentration, pre-induction optical density, and post-induction temperature on the protein expression level content were evaluated. Results: The proposed quadratic model demonstrated a significant effect of the two variables A (time) and C (temperature) on protein synthesis. An inducer concentration of 0.491 mM, the pre-induction optical density (OD600) of 0.8, and a temperature of 23 °C for 23.878 hours were found to be the best growth conditions for high yield Trx-MOG synthesis. The optimum protein concentration was attained (163.96 µg/mL) and was within the range of (200.04 µg/mL), which was the value predicted. Conclusion: The study concluded that RSM optimization effectively increased the production of Trx-MOG in E. coli, which could have the potential for large-scale fermentation.

Oxidative stress changes the effectiveness of artemisinin in Plasmodium falciparum.

Emerging resistance to the frontline antimalarial drug artemisinin represents a significant threat to worldwide malaria control and elimination. The patterns of parasite changes associated with emerging resistance represent a complex array of metabolic processes evident in various genetic mutations and altered transcription profiles. Genetic factors identified in regulating P. falciparum sensitivity to artemisinin overlap with the parasite's responses to malarial fever, sickle trait, and other types of oxidative stresses, suggesting conserved inducible survival responses. In this study we show that intraerythrocytic stress conditions, oxidative stress and heat shock, can significantly decrease the sensitivity of the parasite to artemisinin and lumefantrine, respectively. These results indicate that an intraerythrocytic oxidative stress microenvironment and heat-shock condition can alter antimalarial drug efficacy. Evaluating efficacy of antimalarial drugs under ideal in vitro culture conditions may not accurately predict drug efficacy in all malaria patients.

Abstract A036: Development of a stirred-tank bioreactor scale-up approach to allogeneic natural killer cell culture

Abstract Natural killer (NK) cells are innate lymphocytes that play a critical role in the immune response, as they recognize and eliminate virus-infected, cancerous, and other abnormal cells in an antigen-independent manner. This unique method of recognition makes NK cells an attractive target for utilization as an immunotherapy, as studies have demonstrated they reduce both the induction of graft-versus-host disease and the risk of cytokine release syndrome compared to T-cell based therapies. Additionally, these advantages over canonical T-cell therapies make NK therapies attractive candidates for allogeneic, as opposed to autologous, products. Given their cost-effective scale-up potential, developing a stirred-tank bioreactor (STR)-based NK cell expansion platform to achieve clinical-level expansion is of significant interest, as it would reduce the cost of producing such therapies and improve patient accessibility. To test ideal culture conditions for STR expansion, NK cells were first isolated from healthy donor Leukopaks prior to cryopreservation. Frozen, isolated NK cells were then thawed, co-cultured with varying ratios of irradiated K562 cells and supplemental cytokines from Cytiva in a 6-well G-Rex or DASbox® STR. Finally, cells were harvested for downstream analytics, including identity and functional characterization by flow cytometry and potency assays. A fold expansion of greater than 100 was achieved once the ratio of irradiated K562 to NK cells was optimized. One of the primary challenges encountered was donor-to-donor variation, not only with respect to proliferative capacity, but NK functional phenotype and activity as well. Therefore, a small-scale donor screen was conducted and donors with similar proliferative and functional capabilities were selected for large-scale expansion. Future work will focus on optimizing NK cell transduction to enable genetic customization and scaling-up to a clinically-relevant STR. The successful small-scale NK expansion in an STR demonstrated here lays the groundwork for an expansion platform that enables the manufacturing of various NK-based therapeutics at clinical scale. Citation Format: Mathura Yogalingam, Rebecca Jagroop, Alireza Khalajzadeh, Thergiory Irrazabal, Melissa Perea, Pooja Pushparaj, Roxana Roshankhah, Paul Bowles, Michael Le. Development of a stirred-tank bioreactor scale-up approach to allogeneic natural killer cell culture [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A036.

Survival of Streptococcus equi subsp. zooepidemicus on environmental samples is affected by material type and incubation temperature

Streptococcus equi subsp. zooepidemicus is an opportunistic pathogen associated with disease in a range of domestic and wild animals. Despite its importance, very limited data is available on its survival and persistence on the environment. The goal of this study was to evaluate survival of S. zooepidemicus under ideal culture conditions and farm-like setting, in various surface types. Rubber, plastic, wood, and concrete samples were sterilized and inoculated with 109 CFU/mL of S. zooepidemicus with or without feces, and cultured under ideal conditions (37 °C, 5% CO2) or farm-like settings (20oC on air) for a maximum period of 25 days (n = 3/material/environment/feces-group/time-point). Under ideal conditions without feces, the bacterium survived for up to 17 days on plastic and rubber surfaces, 4 days on wood and less than 1 day on concrete (P < 0.05 between materials). Samples under ideal conditions with feces and farm-like settings without feces were negative by day 1 post-inoculation, regardless of the surface material used. Wood and concrete allowed S. zooepidemicus persistence for up to 3 days under farm-like settings when feces were present. This data suggests that environmental persistence of S. zooepidemicus is affected by surface type and incubation temperature.

Nitrogen removal by Rhodococcus sp. SY24 under linear alkylbenzene sulphonate stress: Carbon source metabolism activity, kinetics, and optimum culture conditions

Artificial intervention combined with stress acclimation was used to screen a heterotrophic nitrifying-aerobic denitrifying (HN-AD) bacterial, strain Rhodococcus SY24, resistant to linear alkylbenzenesulfonic acid (LAS) stress. When LAS was<15 mg/L, strain SY24 performed better cell growth and carbon source metabolism activity. The maximum nitrification and denitrification rates of SY24 under LAS stress could reach 1.18 mg/L/h and 1.05 mg/L/h, respectively, which were 13.80 % and 8.81 % higher than those of the original strain CPZ24. Higher LAS tolerance was seen in the functional genes (amoA, nxrA, napA, narG, nirK, nirS, norB, and nosZ). Response surface modeling revealed that 2 mg/L LAS, sodium succinate as a carbon source, 190 rams, and carbon/nitrogen 11 were the ideal culture conditions for SY24 to nitrogen removal under the LAS environment. This study offered a new screening strategy for the functional species, and strain SY24 showed significant LAS tolerance and HN-AD potential.

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications.

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

Bioprocess optimization of interferon β-1-a in Pichia pastoris and its improved inhibitory effect against hepatocellular carcinoma cells

Interferon-β-1a (INF-β-1a) has gained significant attention due to its emerging applications in the treatment of different human diseases. Therefore, many researchers have attempted to produce it in large quantities and also in a biologically active form using different expression systems. In the present study, we aimed to improve the expression level of INF-β-1a by Pichia pastoris using optimization of culture conditions. The codon-optimized INF-β- 1a gene was cloned into pPICZαA plasmid under the control of alcohol oxidase I (AOX1) promoter. The protein expression was induced using different concentrations of methanol at different pHs and temperatures. The biological activity of produced protein was evaluated by anti-proliferative assay. The ideal culture conditions for the expression of INF-β-1a by P. pastoris were found to be induction with 2% methanol at pH 7.0 culture medium at 30 C which yielded a concentration of 15.5 mg/L INF-β-1a in a shake flask. Our results indicate that differences in glycosylation pattern could result in different biological activities as INF- β-1a produced by P. pastoris could significantly more reduce the cell viability of HepG-2 cells, a hepatocellular carcinoma cell line, than a commercially available form of this protein produced by CHO.

Comparative analysis of the potential of the secretomes of cardiac resident stromal cells and fibroblasts.

The secretome of different cell types has been applied on in vitro and in vivo assays, indicating considerable therapeutic potential. However, the choice of the ideal cell type and culture conditions for obtaining the best set of soluble factors, as well as the assays to assess specific effects, remain subjects of vigorous debate. In this study, we used mass spectrometry to characterize the secretomes of ventricle derived-cardiac resident stromal cells (vCRSC) and human dermal fibroblasts (HDFs) and evaluate them in an effort to understand the niche specificity of biological responses toward different cellular behaviors, such as cell proliferation, adhesion, migration, and differentiation. It was interesting to note that the HDF and vCRSC secretomes were both able to induce proliferation and cardiac differentiation of H9c2 cells, as well as to increase the adhesion activity of H9c2 cells and human umbilical vein endothelial cells. Analysis of the secretome composition showed that the vCRSCs derived from different donors secreted a similar set of proteins. Despite the differences, almost half of the proteins identified in conditioned medium were common to both HDF and vCRSC. Consequently, a high number of common biological processes were identified in the secretomes of the two cell types, which could help to explain the similar results observed in the in vitro assays. We show that soluble factors secreted by both HDF and vCRSC are able to promote proliferation and differentiation of cardiomyoblasts in vitro. Our study indicates the possible use of vCRSC or HDF secretomes in acellular therapies for regenerative medicine.

IDEAL CULTURE CONDITIONS FOR ENHANCING FULL PREIMPLANTATION DEVELOPMENT OF EMBRYOS GENERATED THROUGH SOMATIC CELL HAPLOIDIZATION

To identify the ideal culture conditions for promoting full preimplantation development of artificial embryos generated through somatic cell hapoloidization (SCH). Metaphase II (MII) oocytes from B6D2F1 mice were enucleated under Oosight™ visualization and exposure to cytochalasin B. Nuclear transfer of an individual FVB mouse cumulus cell was conducted by transfer into the perivitelline space of the resulting ooplast and fused using Sendai virus. Development of a pseudo-meiotic spindle was assessed in the reconstructed oocytes 2 hours after transfer, and the oocytes were then piezo-ICSI inseminated. Zygotes were cultured either in KSOM medium (KSOM), KSOM supplemented with Trichostatin A (TSA), or KSOM supplemented with fasudil, scriptaid, and RS-1 (cocktail). Trichostatin A and scriptaid are both histone-deacetylase inhibitors that may improve cellular reprogramming and embryo development after nuclear transfer. Fasudil, a Rho-associated kinase inhibitor, reduces apoptosis, while RS-1 aids in DNA damage repair. Zygotes were cultured in TSA and cocktail for 24 hrs only, before being changed to KSOM. Intact oocytes were ICSI inseminated as controls and cultured in KSOM (control). All four groups were monitored in a time-lapse imaging system for 96hr. A total of 527 MII oocytes were enucleated, resulting in 522 ooplasts (99.0%). Nuclear transfer of these ooplasts resulted in a survival rate of 99.6%, generating 520 reconstructed oocytes. A positive spindle was assessed in 360 artificial oocytes (69.2%), and piezo-ICSI of these spindle-positive oocytes resulted in a survival rate of 75.0%. ICSI insemination of 60 control oocytes had a comparable survival rate (83.3%). Fertilization rates were similar among all culture conditions, as well as to the control, with rates of 91.2% for the cocktail group, 93.8% for the KSOM group, 94.4% for the TSA group, and 94.0% for the control. The cocktail group exhibited significant delays at all stages (P<0.05) except the 2-cell stage when compared to the control. KSOM-cultured embryos had comparable morphokinetic development to the control up to the blastocyst stage, except for a delay in pronuclei appearance (P<0.002). Morphokinetic parameters of TSA embryos were comparable to the controls at all stages, beginning with the pseudo-second polar body extrusion (haploidization) up until the blastocyst stage. The intact oocyte control had an 82.3% blastulation rate compared to all experimental groups (P<0.00001). The cocktail cohort had a blastulation rate of only 9.6%, and the KSOM group had a blastulation rate of 14.5%; the highest blastulation rate was reached by the TSA group at 23.5%. Although the blastocyst development was still suboptimal, among the culture media tested, the addition of a histone-deacetylase inhibitor (TSA) yielded artificial embryos with morphokinetic parameters similar to the control.

P–804 Morphokinetic development by time-lapse imaging of conceptuses generated from artificial oocytes

Abstract Study question What are the ideal culture conditions to enhance full preimplantation development of embryos generated by FVB somatic cell haploidization (SCH) in the mouse model? Summary answer The presence of a histone deacetylase inhibitor yielded the best morphokinetic development of expanded blastocysts generated by FVB SCH, comparable to control blastocysts. What is known already Various culture conditions and medium supplements have been proposed to promote preimplantation development of embryos generated by SCH, including supplementation with trichostatin A (TSA), fasudil, scriptaid, and RAD–51 stimulatory compound–1 (RS–1). TSA and scriptaid, both histone-deacetylase inhibitors, have been found to improve embryo development following nuclear transfer by enhancing histone acetylation and cellular reprogramming. Additionally, fasudil is a Rho-associated kinase inhibitor that has been shown to reduce apoptosis and promote cell proliferation. Finally, RS–1 stimulates RAD51 activity, which promotes the repair of DNA damage and increases the efficacy of somatic cell reprogramming. Study design, size, duration B6D2F1 mouse metaphase II (MII) oocytes underwent enucleation and nuclear transfer, or were ICSI inseminated serving as controls. Reconstituted oocytes showing development of a meiotic-like spindle demonstrated successful SCH, and were ICSI inseminated. SCH conceptuses were cultured in one of three groups: KSOM, KSOM supplemented with TSA (TSA), or KSOM supplemented with fasudil, scriptaid, and RS–1 (Cocktail). ICSI controls (ICSIC) were cultured in KSOM medium. Fertilization and full preimplantation development were compared among all groups. Participants/materials, setting, methods Ooplasts were generated from MII oocytes by removing spindle complexes under OosightÔ visualization and cytochalasin B exposure. A single FVB mouse cumulus cell was transferred into the perivitelline space and fused with the ooplast, facilitated by Sendai virus. Reconstructed oocytes with novel pseudo-meiotic spindles underwent piezo-ICSI and were cultured in different media conditions in a time-lapse imaging system up to 96h. TSA and Cocktail embryos had media changed to regular KSOM 10 hours after insemination. Main results and the role of chance A total of 274 B6D2F1 MII oocytes were enucleated, resulting in a 95.9% survival rate. All ooplasts survived nuclear transfer and 62.1% successfully haploidized after 2 hours. ICSIC and reconstituted SCH oocytes survived piezo-ICSI at rates of 81.5% and 57.0%, respectively (P &amp;lt; 0.01). SCH embryos were then allocated into KSOM, TSA supplied, and Cocktail media. Fertilization rates for ICSIC, KSOM, and TSA embryos were 92.4%, 90.7%, and 94.4%, respectively, while the rate for embryos cultured in Cocktail was only 71.9% (P &amp;lt; 0.03). While embryos cultured in Cocktail had a comparable 2-cell timing to ICSIC, embryos in TSA reached developmental milestones with a closer timing to the ICSIC, having minor delays at the 3-, 4-, and 6-cell stages (P &amp;lt; 0.05). KSOM- and Cocktail-cultured embryos were delayed at most of the stages (P &amp;lt; 0.01), except for the two-pronuclei appearance. Although the TSA group displayed the best embryo developmental pattern, the final rate of blastocyst development was somewhat homogeneous with rates of 15.4%, 23.5%, and 13.0% for the KSOM, TSA, and Cocktail groups, respectively (P &amp;lt; 0.001), and remarkably lower than the ICSIC (81.6%). Limitations, reasons for caution Although live pups have been obtained using BDF cumulus cells, embryos generated by FVB cumulus cells show a remarkably lower blastocyst development, but maintain morphokinetic characteristics similar to ICSIC in the presence of TSA. Wider implications of the findings: While using different strains to enhance genetic variance, the morphokinetic analysis of preimplantation embryos in ideal culture conditions is paramount to the progress of neogametogenesis. The implementation of this technique may soon help create genotyped oocytes for women with compromised ovarian reserve. Trial registration number N/A

Mesenchymal Stem Cell-Based Therapy as an Alternative to the Treatment of Acute Respiratory Distress Syndrome: Current Evidence and Future Perspectives.

Acute respiratory distress syndrome (ARDS) represents a current challenge for medicine due to its incidence, morbidity and mortality and, also, the absence of an optimal treatment. The COVID-19 outbreak only increased the urgent demand for an affordable, safe and effective treatment for this process. Early clinical trials suggest the therapeutic usefulness of mesenchymal stem cells (MSCs) in acute lung injury (ALI) and ARDS. MSC-based therapies show antimicrobial, anti-inflammatory, regenerative, angiogenic, antifibrotic, anti-oxidative stress and anti-apoptotic actions, which can thwart the physiopathological mechanisms engaged in ARDS. In addition, MSC secretome and their derived products, especially exosomes, may reproduce the therapeutic effects of MSC in lung injury. This last strategy of treatment could avoid several safety issues potentially associated with the transplantation of living and proliferative cell populations and may be formulated in different forms. However, the following diverse limitations must be addressed: (i) selection of the optimal MSC, bearing in mind both the heterogeneity among donors and across different histological origins, (ii) massive obtention of these biological products through genetic manipulations of the most appropriate MSC, (iii) bioreactors that allow their growth in 3D, (iv) ideal culture conditions and (v) adequate functional testing of these obtaining biological products before their clinical application.

PHOTOBIOMODULATORY EFFECT OF LOW-INTENSITY LASER RADIATION ON MULTICELLULAR SPHEROIDS

Photobiomodulatory effects of low-intensity laser radiation (LILR) in cells cultured in standard, two-dimensional conditions are well established. Conversely, the characteristics of this effect in three-dimensional (3D) cultures, which are currently recommended due to the greater similarity with cellular behavior in vivo, have not yet been widely investigated. The objective of this work was to analyze the biomodulator effect of LILR, on the wavelength (λ) of 685 nm, on the constitution process and on the viability of cells cultured as multicellular spheroid (MSs). For this, agarose molds containing microwells were seeded (2x105 cell/ml) with osteogenic precursor cells (OPCs - MC3T3-E1) and kept under ideal culture conditions. The molds were irradiated for five consecutive days with doses of 0.5, 1.0 and 1.5 J/cm², the first irradiation being performed immediately after sowing. The process of constitution of MSs was analyzed and the cultures were submitted to the cell viability test. The results demonstrated that the LILR at λ 685 nm exerted a dose-dependent biomodulatory effect on cell metabolism and on the process of constituting the MSs of OPCs. These results demonstrate the potential of photobiomodulation to contribute to the process of constituting MSs, which can be explored in the strategies of multicellular spheroids therapy used in regenerative medicine and bioprinting.

Poly-(lactic acid) and fibrin bioactive cellularized scaffold for use in bone regenerative medicine: Proof of concept

Bone regenerative medicine (BRM) aims to overcome the limitations of conventional treatments for critical bone defects by developing therapeutic strategies, based on temporary bioactive substitutes, capable of stimulating, sustaining, and guiding tissue regeneration. The aim of this study was to validate the “proof of concept” of a cellularized bioactive scaffold and establish its potential for use in BRM. For this purpose, three-dimensional scaffolds of poly-(lactic acid) (PLA), produced by the additive manufacturing technique, were incorporated into a human platelet-rich plasma (PRP-h) fibrin matrix containing human infrapatellar fat pad mesenchymal stem cells (hIFPMSC). The scaffolds (PLA/finbrin-bioactive) were kept under ideal culture conditions in a medium free from fetal bovine serum and analyzed at 5 and 10 days by Scanning Electron Microscopy (SEM), Fourrier Transform Infrared (FTIR), Circular Dichroism and fluorescence microscopy. The results demonstrated the feasibility of obtaining a rigid, cytocompatible, and cellularized three-dimensional structure. In addition, PRP platelets and leukocytes were able to provide a bioactive environment capable of maintaining the viability of hIFPMSC into scaffolds. The results validate the concept of a customizable, bioactive, cellularized, and non-immunogenic strategy for application in BRM.

Capture, Storage and Utilization of Carbon Dioxide by Microalgae and Production of Biomaterials

Abstract Carbon dioxide emissions are strongly related to climate change and increase of global temperature. Whilst a complete change in producing materials and energy and in traffic and transportation systems is already in progress and circular economy concepts are on working, Carbon Capture and Storage (CCS) and Carbon Capture and Utilisation (CCU) represent technically practicable operative strategies. Both technologies have main challenges related to high costs, so that further advanced research is required to obtain feasible options. In this article, the focus is mainly on CCU using microalgae that are able to use CO2 as building block for value-added products such as biofuels, EPS (Extracellular Polymeric Substances), biomaterials and electricity. The results of three strains (UTEX 90, CC 2656, and CC 1010) of the microalgal organism Chlamydomonas reinhardtii are discussed. The results about ideal culture conditions suggest incubation temperature of 30 °C, pH between 6.5 and 7.0, concentrations of acetate between 1.6 and 2.3 g L–1 and of ammonium chloride between 0.1 and 0.5 g L–1, the addition of glucose This green microalga is a valid model system to optimize the production of biomass, carbohydrates and lipids.

A practical guide to microfabrication and patterning of hydrogels for biomimetic cell culture scaffolds

This review article describes microfabrication techniques to define chemical, mechanical and structural patterns in hydrogels and how these can be used to prepare in vivo like, i.e. biomimetic, cell culture scaffolds. Hydrogels are attractive materials for 3D cell cultures as they provide ideal culture conditions and they are becoming more prominently used. Single material gels without any modifications do however have their limitation in use and much can be gained by in improving the in vivo resemblance of simple hydrogel cell culture scaffolds. This review article discusses the most commonly used cross-linking strategies used for hydrogel-based culture scaffolds and gives a brief introduction to microfabrication methods that can be used to define chemical, mechanical and structural patterns in hydrogels with micrometre resolution. The review article also describes a selection of literature references using these microfabrication techniques to prepare organ and disease models with controlled cell adhesion, proliferation and migration. It is intended to serve as an introduction to microfabrication of hydrogels and an inspiration for novel interdisciplinary research projects.

Characterization and population dynamics of germ cells in adult macaque testicular cultures.

BackgroundFrom a biological and clinical perspective, it is imperative to establish primate spermatogonial cultures. Due to limited availability of human testicular tissues, the macaque (Macaca fascicularis) was employed as non-human primate model. The aim of this study was to characterize the expression of somatic as well as germ cell markers in testicular tissues and to establish macaque testicular primary cell cultures.Materials and methodsCharacterization of macaque testicular cell population was performed by immunohistochemical analyses for somatic cell markers (SOX9, VIM, SMA) as well as for germ cell markers (UTF1, MAGEA4, VASA). Testicular cells from adult macaque testes (n = 4) were isolated and cultured for 21 days using three stem cell culture media (SSC, PS and SM). An extended marker gene panel (SOX9, VIM, ACTA2; UTF1, FGFR3, MAGEA4, BOLL, DDX4) was then employed to assess the changes in gene expression levels and throughout the in vitro culture period. Dynamics of the spermatogonial population was further investigated by quantitative analysis of immunofluorescence-labeled MAGEA4-positive cells (n = 3).ResultsRNA expression analyses of cell cultures revealed that parallel to decreasing SOX9-expressing Sertoli cells, maintenance of VIM and ACTA2-expressing somatic cells was observed. Expression levels of germ cell marker genes UTF1, FGFR3 and MAGEA4 were maintained until day 14 in SSC and SM media. Findings from MAGEA4 immunofluorescence staining corroborate mRNA expression profiling and substantiate the overall maintenance of MAGEA4-positive pre- and early meiotic germ cells until day 14.ConclusionsOur findings demonstrate maintenance of macaque germ cell subpopulations in vitro. This study provides novel perspective and proof that macaques could be used as a research model for establishing in vitro germ cell-somatic cell cultures, to identify ideal culture conditions for long-term maintenance of primate germ cell subpopulation in vitro.

Investigating the Potential of Ion Mobility-Mass Spectrometry for Microalgae Biomass Characterization.

Algae biomass is formed by an extremely complex set of metabolites, and its molecular characterization has been very challenging. We report the characterization of microalgae extracts via traveling wave ion mobility-mass spectrometry (TWIM-MS) by two different analysis strategies. First, the extracts were analyzed by direct infusion electrospray ionization (ESI) with no previous chromatographic separation (DI-ESI-TWIM-MS). Second, the samples were screened for metabolites and lipids using an untargeted high-throughput method that employs ultrahigh-performance liquid chromatography (UHPLC) using data-independent analysis (DIA) - MSE (UHPLC-HDMSE). Sixteen different microalgae biomasses were evaluated by both strategies. DI-ESI-TWIM-MS was able, via distinct drift times, to set apart different classes of metabolites, with the differences in the profiles of each microalga readily evident. With the UHPLC-HDMSE approach, 1251 different compounds were putatively annotated across 16 samples with 210 classified as lipids. From the normalized abundance for each annotated compound category, a detailed profiling in terms of metabolites, lipids, and lipid classes of each sample was performed. The reported workflow represents a powerful tool to determine the most suitable biotechnological applications for a given alga type and may allow for real-time monitoring of the algae composition distribution as a function of growth conditions, feedstocks, and the like. The determination of collision cross section results in improved confidence in the identification of triacylglycerols in samples, highly applicable to biofuels production. The two analysis strategies explored in this work offer powerful tools for the biomass industry by aiding in the identification of ideal strains and culture conditions for a specific application, saving analysis time and facilitating identification of a large number of constituents at once.