Cell Bank Research Articles

Potential and Reality of Cryopreserving Somatic Cells of Wild Felids for Conservation

The loss of biodiversity caused by anthropogenic actions is also a reality for the members of the Felidae family. Except for the domestic cat, all felid species have some degree of threat of extinction in their natural habitat. For this reason, felids have been included in conservation-related studies. This scenario has aroused increasing interest in the formation of somatic cell banks, which when efficiently implemented can be used in preservation strategies for the species. Nevertheless, one of the important steps in the formation of these banks is the understanding of the technical principles and variations involved in cryopreservation techniques, especially because cryopreservation increases the possibilities for Assisted Reproduction Technologies (ARTs) by making the use of biological materials independent of time and space. In wild felids, several species already have promising results in the formation of somatic cell banks, and studies aimed at better viability rates have been constantly proposed, as well as new species have been studied. In some species, aspects involved in successful cryopreservation are already well defined, and slow freezing associated with cryoprotectant solutions composed of intra- and extracellular substances is the most useful approach. The aim of this review was to present the main parameters involved in the elaboration of a somatic cell cryopreservation protocol and their effects, as well as to address the main results achieved for different wild felids.

A Review: Methodologies to Promote the Differentiation of Mesenchymal Stem Cells for the Regeneration of Intervertebral Disc Cells Following Intervertebral Disc Degeneration.

Intervertebral disc (IVD) degeneration (IDD), a highly prevalent pathological condition worldwide, is widely associated with back pain. Treatments available compensate for the impaired function of the degenerated IVD but typically have incomplete resolutions because of their adverse complications. Therefore, fundamental regenerative treatments need exploration. Mesenchymal stem cell (MSC) therapy has been recognized as a mainstream research objective by the World Health Organization and was consequently studied by various research groups. Implanted MSCs exert anti-inflammatory, anti-apoptotic, and anti-pyroptotic effects and promote extracellular component production, as well as differentiation into IVD cells themselves. Hence, the ultimate goal of MSC therapy is to recover IVD cells and consequently regenerate the extracellular matrix of degenerated IVDs. Notably, in addition to MSC implantation, healthy nucleus pulposus (NP) cells (NPCs) have been implanted to regenerate NP, which is currently undergoing clinical trials. NPC-derived exosomes have been investigated for their ability to differentiate MSCs from NPC-like phenotypes. A stable and economical source of IVD cells may include allogeneic MSCs from the cell bank for differentiation into IVD cells. Therefore, multiple alternative therapeutic options should be considered if a refined protocol for the differentiation of MSCs into IVD cells is established. In this study, we comprehensively reviewed the molecules, scaffolds, and environmental factors that facilitate the differentiation of MSCs into IVD cells for regenerative therapies for IDD.

Novel anti-CD30/CD3 bispecific antibodies activate human T cells and mediate potent anti-tumor activity.

CD30 is expressed on Hodgkin lymphomas (HL), many non-Hodgkin lymphomas (NHLs), and non-lymphoid malignancies in children and adults. Tumor expression, combined with restricted expression in healthy tissues, identifies CD30 as a promising immunotherapy target. An anti-CD30 antibody-drug conjugate (ADC) has been approved by the FDA for HL. While anti-CD30 ADCs and chimeric antigen receptors (CARs) have shown promise, their shortcomings and toxicities suggest that alternative treatments are needed. We developed novel anti-CD30 x anti-CD3 bispecific antibodies (biAbs) to coat activated patient T cells (ATCs) ex vivo prior to autologous re-infusions. Our goal is to harness the dual specificity of the biAb, the power of cellular therapy, and the safety of non-genetically modified autologous T cell infusions. We present a comprehensive characterization of the CD30 binding and tumor cell killing properties of these biAbs. Five unique murine monoclonal antibodies (mAbs) were generated against the extracellular domain of human CD30. Resultant anti-CD30 mAbs were purified and screened for binding specificity, affinity, and epitope recognition. Two lead mAb candidates with unique sequences and CD30 binding clusters that differ from the ADC in clinical use were identified. These mAbs were chemically conjugated with OKT3 (an anti-CD3 mAb). ATCs were armed and evaluated in vitro for binding, cytokine production, and cytotoxicity against tumor lines and then in vivo for tumor cell killing. Our lead mAb was subcloned to make a Master Cell Bank (MCB) and screened for binding against a library of human cell surface proteins. Only huCD30 was bound. These studies support a clinical trial in development employing ex vivo-loading of autologous T cells with this novel biAb.

Cost-Effective Cultivation of Native PGPB Sinorhizobium Strains in a Homemade Bioreactor for Enhanced Plant Growth.

The implementation of bioreactor systems for the production of bacterial inoculants as biofertilizers has become very important in recent decades. However, it is essential to know the bacterial growth optimal conditions to optimize the production and efficiency of bioinoculants. The aim of this work was to identify the best nutriment and mixing conditions to improve the specific cell growth rates (µ) of two PGPB (plant growth-promoting bacteria) rhizobial strains at the bioreactor level. For this purpose, the strains Sinorhizobium mexicanum ITTG-R7T and Sinorhizobium chiapanecum ITTG-S70T were previously reactivated in a PY-Ca2+ (peptone casein, yeast extract, and calcium) culture medium. Afterward, a master cell bank (MCB) was made in order to maintain the viability and quality of the strains. The kinetic characterization of each bacterial strain was carried out in s shaken flask. Then, the effect of the carbon and nitrogen sources and mechanical agitation was evaluated through a factorial design and response surface methodology (RSM) for cell growth optimization, where µ was considered a response variable. The efficiency of biomass production was determined in a homemade bioreactor, taking into account the optimal conditions obtained during the experiment conducted at the shaken flask stage. In order to evaluate the biological quality of the product obtained in the bioreactor, the bacterial strains were inoculated in common bean (Phaseolus vulgaris var. Jamapa) plants under bioclimatic chamber conditions. The maximum cell growth rate in both PGPB strains was obtained using a Y-Ca2+ (yeast extract and calcium) medium and stirred at 200 and 300 rpm. Under these growth conditions, the Sinorhizobium strains exhibited a high nitrogen-fixing capacity, which had a significant (p < 0.05) impact on the growth of the test plants. The bioreactor system was found to be an efficient alternative for the large-scale production of PGPB rhizobial bacteria, which are intended for use as biofertilizers in agriculture.

A review of Innovation in Cell Banking, Expansion, and Production Culture

A review of Innovation in Cell Banking, Expansion, and Production Culture

From frozen cell bank to product assay: high-throughput strain characterisation for autonomous Design-Build-Test-Learn cycles

BackgroundModern genome editing enables rapid construction of genetic variants, which are further developed in Design-Build-Test-Learn cycles. To operate such cycles in high throughput, fully automated screening, including cultivation and analytics, is crucial in the Test phase. Here, we present the required steps to meet these demands, resulting in an automated microbioreactor platform that facilitates autonomous phenotyping from cryo culture to product assay.ResultsFirst, an automated deep freezer was integrated into the robotic platform to provide working cell banks at all times. A mobile cart allows flexible docking of the freezer to multiple platforms. Next, precultures were integrated within the microtiter plate for cultivation, resulting in highly reproducible main cultures as demonstrated for Corynebacterium glutamicum. To avoid manual exchange of microtiter plates after cultivation, two clean-in-place strategies were established and validated, resulting in restored sterile conditions within two hours. Combined with the previous steps, these changes enable a flexible start of experiments and greatly increase the walk-away time.ConclusionsOverall, this work demonstrates the capability of our microbioreactor platform to perform autonomous, consecutive cultivation and phenotyping experiments. As highlighted in a case study of cutinase-secreting strains of C. glutamicum, the new procedure allows for flexible experimentation without human interaction while maintaining high reproducibility in early-stage screening processes.

Dead but functional liquid nitrogen‐treated umbilical cord mesenchymal stem cells accelerate full‐thickness skin wound healing

AbstractBackgroundThe direct application of umbilical cord‐derived mesenchymal stem cells (UCMSCs) for promoting skin wound healing and regeneration is challenging due to strict maintenance requirements and unpredictable differentiation results.MethodsWe developed dead but functional liquid nitrogen‐treated UCMSCs (LNT‐MSCs) by rapidly immersing live UCMSCs (live‐MSCs) in liquid nitrogen.ResultsThe LNT‐MSCs maintained similar cellular structures and surface markers to those of live‐MSCs. We evaluated the therapeutic effects of both live‐MSCs and LNT‐MSCs on full‐thickness skin wound healing in rats. Our results showed that the LNT‐MSCs accelerated wound closure by enhancing the proliferation and migration of skin cells, promoting angiogenesis, and inducing a favourable macrophage phenotype shift. The regenerative healing effect of LNT‐MSCs was comparable to that of live‐MSCs, making them a potential alternative strategy for accelerating wound closure that avoids unpredictable differentiation results and creates a ready‐to‐use cell bank for clinical applications.

Examining the Effect of Extract of Oak Fruit Jaft on AGS Cell Lines

Background: Gastrointestinal carcinoma comprises 5% of all pediatric cancer in children. Given that the possible and beneficial effect of the Jaft extract in the treatment of gastric cancer is not known and there is no comprehensive study in this regard, this study aimed to assess the effect of Jaft extract on gastric cancer cell lines.&#x0D; Materials and Methods: In this case-control study, oak fruit was collected from the mountains of Lorestan province. A gastric cancer (AGS) cell line was obtained from the Institute Pasteur cell bank and was cultured. After the preparation of the ethanolic extract of Jaft, the cell viability of the gastric cancer cells treated with Jaft extract was investigated by MTT assay. Quantitative Reverse Transcription PCR was used for assessing the expression of BAX, and BCL2 genes.&#x0D; Results: The half maximal inhibitory concentration (IC50) value of Jaft extract was 162 µg/ml. The BAX gene expression was different between the case and control groups. In this regard, the expression of the BAX gene was increased in the concentration of 162 (P&lt;0.01) and 250 µg/ml (P&lt;0.001) of Jaft extract compared to the control group. The BCL2 expression was different between the two groups (P&lt;0.05). In this regard, the expression of the BCL2 gene was decreased in the concentration of 162 and 250 µg/ml of Jaft extract compared to the control group.&#x0D; Conclusion: It was found that Jaft extract increased the apoptosis of gastric cancer cells; therefore, it seems that the hydroalchoholic extract of Jaft is an appropriate anticancer medication.

Cardiac Regeneration Using Pluripotent Stem Cells and Controlling Immune Responses.

Pluripotent stem cell (PSC)-derived cardiomyocytes are a promising source of cells in myocardial regeneration therapy for end-stage heart failure. Because most previous reports have focussed on xenotransplantation models using immunocompromised animals, studies on immune rejection in allogeneic transplantation models are needed for preclinical and clinical applications. Human leukocyte antigen (HLA) plays an important role in allogeneic transplantation, and cell bank projects are currently underway worldwide to stock induced pluripotent stem cells (iPSCs) generated from healthy individuals with homozygous HLA haplotypes. However, it is difficult to stock iPSCs that match the entire population in these cell banks; thus, several groups have produced hypoimmunogenic PSCs by knocking out HLA. These HLA-knockout PSCs were able to avoid rejection by T cells but still suffered rejection by natural killer(NK) cells caused by 'missing self-recognition'. Recent studies have attempted to generate hypoimmunogenic PSCs with gene editing to inhibit NK cell activation. Regenerative medicine using autologous iPSCs can be an ideal transplantation therapy, but, currently, there are major hurdles to its practical application. Hopefully, further research will resolve these issues. This review provides an overview of the current understanding and progress in this field.

의약품 안정성시험 및 바이오의약품 분석지원센터 구축 연구

This study was undertaken to suggest the necessity of establishing stability test and biopharmaceutical analysis support center. Feasibility study on center establishment was performed as online survey to factory manager and person in charge in 39 pharmaceutical companies in Korea. More than seventy percent of 39 companies responded that they need to establish it. Eighty percent of respondents answered that they would entrust the entire test items of stability test and 71.8% of respondents answered that they would entrust biopharmaceutical cell bank and protein analysis. The construction cost of two centers with 2 stories and 1 basement floor and 3000m2 was 6.37 billion won. The stability test center need to 173 EA of 71 types of equipment and 4.086 billion won in budget for equipment purchases. The biopharmaceutical analysis support center need to 169 EA of 77 types of equipment and about 7.2 billion won in budget for equipment purchase. Total cost for construction and equipment purchase was 17.65 billion won. The establishment of two centers with expertise and efficiency would provide the competitiveness of the pharmaceutical industry in Korea.

Establishment and characterization of two novel patient-derived cell lines from giant cell tumor of bone.

Giant cell tumor of bone (GCTB) is a rare bone tumor with osteolytic features, composed of stromal cells with a monotonous appearance, macrophages, and osteoclast-like giant cells. GCTB is commonly associated with a pathogenic mutation in the H3-3A gene. While complete surgical resection is the standard cure for GCTB, it often results in local recurrence and, rarely, metastasis. Thus, an effective multidisciplinary treatment approach is necessary. Although patient-derived cell lines is an essential tool for investigating novel treatment strategies, there are only four GCTB cell lines available in public cell banks. Therefore, this study aimed to establish novel GCTB cell lines and successfully created NCC-GCTB6-C1 and NCC-GCTB7-C1 cell lines from two patients' surgically removed tumor tissues. These cell lines exhibited H3-3A gene mutations, consistent proliferation, and invasive properties. After characterizing their behaviors, we performed high-throughput screening of 214 anti-cancer drugs for NCC-GCTB6-C1 and NCC-GCTB7-C1 and integrated their screening data with those of NCC-GCTB1-C1, NCC-GCTB2-C1, NCC-GCTB3-C1, NCC-GCTB4-C1, and NCC-GCTB5-C1 that we previously established. We identified histone deacetylase inhibitor romidepsin as a possible treatment for GCTB. These findings suggest that NCC-GCTB6-C1 and NCC-GCTB7-C1 could be valuable tools for preclinical and basic research on GCTB.

Maximizing the potential for living cell banks to contribute to global conservation priorities.

Although cryobanking represents a powerful conservation tool, a lack of standardized information on the species represented in global cryobanks, and inconsistent prioritization of species for future sampling, hinder the conservation potential of cryobanking, resulting in missed conservation opportunities. We analyze the representation of amphibian, bird, mammal, and reptile species within the San Diego Zoo Wildlife Alliance Frozen Zoo® living cell collection (as of April 2019) and implement a qualitative framework for the prioritization of species for future sampling. We use global conservation assessment schemes (including the International Union for Conservation of Nature (IUCN) Red List of Threatened Species™, the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), the Alliance for Zero Extinction, the EDGE of Existence, and Climate Change Vulnerability), and opportunities for sample acquisition from the global zoo and aquarium community, to identify priority species for cryobanking. We show that 965 species, including 5% of all IUCN Red List "Threatened" amphibians, birds, mammals, and reptiles, were represented in the collection and that sampling from within existing zoo and aquarium collections could increase representation to 16.6% (by sampling an additional 707 "Threatened" species). High-priority species for future cryobanking efforts include the whooping crane (Grus americana), crested ibis (Nipponia nippon), and Siberian crane (Leucogeranus leucogeranus). Each of these species are listed under every conservation assessment scheme and have ex situ populations available for sampling. We also provide species prioritizations based on subsets of these assessment schemes together with sampling opportunities from the global zoo and aquarium community. We highlight the difficulties in obtaining in situ samples, and encourage the formation of a global cryobanking database together with the establishment of new cryobanks in biodiversity-rich regions.

Cytotoxicity of PLGA-zinc oxide nanocomposite on human gingival fibroblasts.

Polylactic-co-glycolic acid and zinc oxide (PLGA-ZnO) nanocomposite has been investigated for its antibacterial properties, which could be beneficial for adding to wound dressings after periodontal surgery. However, its cytotoxicity against human gingival fibroblasts (HGFs) remains unclear and should be evaluated. ZnO nanoparticles were synthesized using the hydrothermal method. These metallic nanoparticles were incorporated into the PLGA matrix by the solvent/non-solvent process. The nanomaterial was evaluated by field emission scanning electron microscopy (FESEM), Fourier transform infrared (FTIR), thermogravimetric analysis (TGA), and x-ray diffraction (XRD) analyses. HGF cells were acquired from the National Cell Bank and categorized into four groups: ZnO, PLGA, ZnO-PLGA, and control. The cells were exposed to different ZnO (1, 20, 40, 60, 80, and 100 µg/mL) and PLGA (0.2, 4, 8, 12, 16, and 20 µg/mL) concentrations for 24 and 48 hours. The cytotoxicity was tested using the MTT assay. The data were analyzed using SPSS 25, and P<0.05 was considered statistically significant. ZnO nanoparticles exhibited significant toxicity at≥40 µg/mL concentrations after 24 hours. Cell viability decreased significantly at all the tested concentrations after 48 hours of exposure. PLGA-ZnO cell viability in 24 hours was similar to the control group for all the concentrations up to 80 µg/mL. ZnO nanoparticles could be toxic against HGF in high concentrations and with prolonged exposure. Therefore, incorporating ZnO nanoparticles into a biocompatible polymer such as PLGA could be a beneficial strategy for reducing their toxicity.

The ELiPSE-1 study: A phase 1, multicenter, open-label study of CNTY-101 in subjects with relapsed or refractory CD19-positive B-cell malignancies.

TPS7580 Background: Century Therapeutics’ foundational technology is built on induced pluripotent stem cells, differentiated from allogeneic approaches that utilize non-renewable donor-derived cells. This approach allows production of a clonal cell bank of precisely edited cells that can be expanded and differentiated to supply large amounts of homogeneous allogeneic therapeutic products for off-the-shelf use. CNTY-101 is an allogeneic Chimeric Antigen Receptor NK (CAR-NK) cell product with genetic modifications designed to specifically target CD19-expressing B-cell malignancies, reduce allorejection in the recipient allowing for repeat dosing, increase persistence, and enable cell elimination in the event of unexpected adverse events. CNTY-101, as a CAR-NK product, has the potential for improved safety over T-cell based cellular therapies. Methods: This first-in-human study will determine the MTD and recommended Phase 2 regimen (RP2R) of CNTY-101 in combination with subcutaneous (SC) IL-2 in patients with R/R aggressive and indolent CD19-positive B-cell Non-Hodgkin lymphomas including diffuse large B-cell lymphoma, mantle cell lymphoma, primary mediastinal large B-cell lymphoma, follicular lymphoma and marginal zone lymphoma, who are without other treatment options. Part 1 of the study (dose escalation using the BOIN design) will start with a dose of CNTY-101 cells without IL-2 (1 to 3 subjects), followed by dose escalation up to four dose cohorts administering a single dose of CNTY-101 cells in combination with IL-2 daily for 8 days (Sched A). After the single-dose MTD (or the maximal single dose) has been reached, a fractionated schedule of 3 doses per 28-day cycle, administered on Days 1, 8, and 15 (Sched B), will also be explored, starting with approximately one-third the maximum single dose level of CNTY-101, followed by escalation/de-escalation. Subjects in whom CNTY-101 shows signs of clinical benefit (i.e. stable disease or better per Lugano 2014 criteria) may receive additional cycles of CNTY-101 if the Investigator, Sponsor, and appropriate regulatory authorities, as required, concur that the benefit-risk is positive. One overall CNTY-101 RP2R will be declared in Part 1. Part 2, expansion, will further evaluate the safety, pharmacokinetics (PK), and efficacy of the CNTY-101 regimen, treating up to an additional 12 subjects to reach a total of approximately 20 subjects treated at the RP2R. In addition to safety and efficacy endpoints, the trial will evaluate exploratory PK, immunogenicity and pharmacodynamic endpoints including evaluations of tumor cfDNA, iNK tumor trafficking and serum cytokines. Clinical trial information: NCT05336409 .

Fresh or Thawed T Cells?: How Modes of Bioavailability Matter in the Study of Translational Medicine

This article explores how cell immunotherapy becomes deliverable at the in-betweens of the bench, bedside, and community in translational medicine. During ethnographic research in an advanced cell therapies unit, I observed how the staff made virus-specific T cells available for therapeutic use. In dialogue with recent Science and Technology Studies scholars’ thinking around the concept of readiness, this paper highlights the analytic richness of paying empirical attention to how biological therapeutics are made available (i.e., their modes of bioavailability). This study identifies two modes of bioavailability for landing cellular drugs at the bedside. In bioavailability mode I, cells are banked in potential donors’ bodies and extracted for fresh infusion. In bioavailability mode II, cells are expanded and cryobanked in a cell bank for thawed infusion. Each mode offers different degrees of readiness and matching. I argue that ethnographic analysis of modes of bioavailability in health innovation and therapeutics implementation enables a less finalist and mass production understanding of translational medicine. This work highlights the relevance of exploring the practices, logistics, and logics that make some modes of bioavailability more valuable than others in pharmaceutical terms—and biomedicine more broadly.

Role and prospect of regenerative medicine in early treatment of combat trauma

The regenerative medicine has made great breakthrough in the repair of combat trauma, showing broad prospects, while the method of regenerative medicine applied in the early treatment of combat trauma is not yet clear. The early treatment of combat trauma includes strict control of bleeding, a large amount of blood transfusion, alleviation of acidosis, and correction of hypothermia and improvement of coagulation dysfunction, etc. This paper focuses on the bio-engineered blood, research and development of homeostatic materials, control of inflammation/infection, regulation of immunity, protection of important organs, establishment of military medical model, research and development of biosensors and drugs, and preventive application of stem cell bank in regenerative and tissue engineering in defense medicine to summarize the role of regenerative medicine in the early treatment of combat trauma, hoping to improve the overall treatment level of combat trauma.

Allele and haplotype frequencies in patients with Hematopoietic stemcell transplantation in National Children’s Hospital

HLA (Human Leukocyte Antigen) gene is located on chromosome 6’s short arm, including several loci. HLA genotyping is necessary for transplantation, especially in hematopoietic stem cell transplantation. A cross-sectional study to determine the frequency of HLA-A, HLA-B, and HLA-DRB1 alleles of 122 patients admitted to the National Children’s Hospital for Hematopoietic stem cell transplantation from the year between 2010 and 2020. The patients’ HLA genotyping was performed by PCR-SSP method using Micro SSPTM Generic HLA Class I and II DNA Typing Tray (One Lambda). The results showed that the HLA-A locus has 12 alleles of which the highest frequency alleles are A*11 (24.18%), A*02 (23.36%), A*33 (20.90%), and A*24 (14.34%). HLA-B locus has 24 alleles in which the highest frequency alleles are B*15 (24.59%), B*58 (12.30%), B*07 (9.02%), and B*46 (8.20%). HLA-C locus has ten alleles of which the highest frequency alleles are C*03 (24.59%), C*07 (22.12%), and C*04 (13.52%). HLA-DRB1 locus has 13 alleles of which the highest frequency alleles are DRB1*12 (23.77%), DRB1*03 (11.07%), DRB1*15 (10.25%), and DRB1*09 &amp; DRB1*07 (8.20%). There are 1984 haplotypes combined from 4 loci HLA-A, HLA-B, HLA-C, and HLA-DRB1, of which the highest frequency haplotypes are A*11/B*15/C*08/DRB1*12 (1.21%); A*33/B*58/C*03/DRB1*03 (1.06%); A*11/B*15/C*04/DRB1*12 (0.76%); A*11/B*15/C*04/DRB1*12 (0.71%), and A*11/B*15/C*03/DRB1*12 (0.71%). The study provides the frequencies of alleles and haplotypes of HLA genotypes in Hematopoietic Stem Cell Transplantation. This is very important information for choosing stem cell donors and establishing the stem cell bank.

Investment in Healthcare through Dental Stem Cell Banking in India

Tooth Banking is a simple alternative to recover stem cells with the potential for differentiation into various cells of the body. Young and healthy pulp is a enriched source of stem cells. These cells can be preserved for a longer time to treat various ailments like heart disease, cancer, repairing or even growing damaged organs etc. Likewise, stem cells recovered can be used in regenerative procedures in dentistry. There are many companies offering tooth banking with simple protocols for stem cell banking and storage. This review is based on various dental stem cell banks and details of harvesting with the applications and limitations of procedures. Sources of information are various articles on stem cell banking from reputed journals. Likewise, informative survey of official websites of dental stem cell banks and verbal information extracted from their representatives.

A pumpless monolayer microfluidic device based on mesenchymal stem cell-conditioned medium promotes neonatal mouse in vitro spermatogenesis

BackgroundChildhood cancer treatment-induced gonadotoxicity causes permanent infertility/sub-infertility in nearly half of males. The current clinical and experimental approaches are limited to cryopreservation of prepubertal testicular strips and in vitro spermatogenesis which are inadequate to achieve the expanded spermatogonial stem/progenitor cells and spermatogenesis in vitro. Recently, we reported the supportive effect of bone marrow-derived mesenchymal cell co-culture which is inadequate after 14 days of culture in static conditions in prepubertal mouse testis due to lack of microvascular flow and diffusion. Therefore, we generated a novel, pumpless, single polydimethylsiloxane-layered testis-on-chip platform providing a continuous and stabilized microfluidic flow and real-time cellular paracrine contribution of allogeneic bone marrow-derived mesenchymal stem cells.MethodsWe aimed to evaluate the efficacy of this new setup in terms of self-renewal of stem/progenitor cells, spermatogenesis and structural and functional maturation of seminiferous tubules in vitro by measuring the number of undifferentiated and differentiating spermatogonia, spermatocytes, spermatids and tubular growth by histochemical, immunohistochemical, flow cytometric and chromatographic techniques.ResultsBone marrow-derived mesenchymal stem cell-based testis-on-chip platform supported the maintenance of SALL4(+) and PLZF(+) spermatogonial stem/progenitor cells, for 42 days. The new setup improved in vitro spermatogenesis in terms of c-Kit(+) differentiating spermatogonia, VASA(+) total germ cells, the meiotic cells including spermatocytes and spermatids and testicular maturation by increasing testosterone concentration and improved tubular growth for 42 days in comparison with hanging drop and non-mesenchymal stem cell control.ConclusionsFuture fertility preservation for male pediatric cancer survivors depends on the protection/expansion of spermatogonial stem/progenitor cell pool and induction of in vitro spermatogenesis. Our findings demonstrate that a novel bone marrow-derived mesenchymal stem cell-based microfluidic testis-on-chip device supporting the maintenance of stem cells and spermatogenesis in prepubertal mice in vitro. This new, cell therapy-based microfluidic platform may contribute to a safe, precision-based cell and tissue banking protocols for prepubertal fertility restoration in future.

Enhanced Proliferative and Osteogenic Potential of Periodontal Ligament Stromal Cells.

Cell-based therapies using periodontal ligament stromal cells (PDLSC) for periodontal regeneration may represent an alternative source for mesenchymal stromal cells (MSC) to MSC derived from bone marrow (MSC(M)) and adipose tissue (MSC(AT)). We aimed to characterize the osteogenic/periodontal potential of PDLSC in comparison to MSC(M) and MSC(AT). PDLSC were obtained from surgically extracted healthy human third molars, while MSC(M) and MSC(AT) were obtained from a previously established cell bank. Flow cytometry, immunocytochemistry, and cell proliferation analyses provided cellular characteristics from each group. Cells from the three groups presented MSC-like morphology, MSC-related marker expression, and multilineage differentiation capacity (adipogenic, chondrogenic, and osteogenic). In this study, PDLSC expressed osteopontin, osteocalcin, and asporin, while MSC(M) and MSC(AT) did not. Of note, only PDLSC expressed CD146, a marker previously applied to identify PDLSC, and presented higher proliferative potential compared to MSC(M) and MSC(AT). Upon osteogenic induction, PDLSC exhibited higher calcium content and enhanced upregulation of osteogenic/periodontal genes compared to MSC(M) and MSC(AT), such as Runx2, Col1A1 and CEMP-1. However, the alkaline phosphatase activity of PDLSC did not increase. Our findings suggest that PDLSC might be a promising cell source for periodontal regeneration, presenting enhanced proliferative and osteogenic potential compared to MSC(M) and MSC(AT).