Cell Isolation Procedure Research Articles

Isolation Procedure for Rat Pancreatic Ductal Cells.

Isolating pancreatic ductal cells from rats is a critical procedure in pancreatic research, offering valuable insights into pancreatic function, pathology, and potential treatments. The process involves several key steps, beginning with the proper removal of the rat's pancreas, followed by the initiation of the ductal cell isolation procedure. This aims to obtain pure and viable ductal cell populations for further experimentation and analysis.

Bursal Tissue Harvested During Rotator Cuff Repair Contains Viable Mesenchymal Stem Cells

PurposeThe purpose of this study was to evaluate the effect of intra operative ablation on the viability, distribution, phenotype, and potential for culture expansion of bursal cells harvested during arthroscopic rotator cuff surgery. MethodsTissue was collected during primary arthroscopic rotator cuff repair on six healthy, randomly selected patients from a fellowship-trained surgeon’s practice between September 2020 and January 2021. There were 3 females (age 60 ± 8 years) and 3 males (age 61 ± 10 years). At the time of surgery, subacromial bursal tissue was subjected to no ablation, one second, or three seconds of ablation. Tissues were collected by an autograft harvesting system connected to an arthroscopic shaver and a pituitary grasper. Tissue fragments from each condition were sampled for viability testing or cell isolation. A viability kit with confocal microscopy was used to assess live and dead cells. Cell isolation consisted of collagenase digestion or placing tissue fragments onto tissue culture treated plates that induced migration of cells out of the tissue. Cell proliferation rates were monitored and surface markers for mesenchymal stem cells (MSC) and pericytes were analyzed via multi-color flow cytometry. ResultsIncreased ablation time significantly reduced cell viability. The mean percentage of live cells was 55.2 ± 27.2% (range 26-90% live) in the control group, 46.8 ± 23.8% (range 9.6-69.6% p = 0.045) in the short, and 35.5 ± 19% (range 11-54% p = 0.03) in the long ablation group. No significant differences in population doubling level (1.6 ± 0.5 days) and population doubling time (6.7 ± 2.4 days) were observed in cells from any treatment. The surface marker profile indicated an MSC phenotype with absence of a pericyte population. Ablation or cell isolation procedure had no significant effect on the surface marker profile of isolated cells. ConclusionRadiofrequency ablation significantly reduced the overall tissue viability, but had no significant effect on cell proliferation, or expression of surface markers on isolated subacromial bursal cells harvested arthroscopically. Clinical RelevanceAnalysis of the viability and performance of cells harvested after the use of ablation devices using mechanical surgical collection during RCR surgery could further our understanding of subacromial bursal tissue and its potential role in augmenting rotator cuff repair healing.

A Scalable, Off-the-Shelf, and Cost-Effective NK Cell Manufacturing Platform for Developing NK Cell-Based Immunotherapy

Natural killer (NK) cells are innate lymphocytes that play a key role in the control of infection and malignancy. NK cells are potent killers of target cells and act in an MHC-independent manner, enabling their application as allogenic therapy for cancer. Human iPSCs are pluripotent with the ability to self-renew, thus providing an inexhaustible source for deriving NK cells. With a two-tiered cell bank system, we can manufacture NK cells with more homogeneity between batches. In principle, one single iPSC clone-derived master cell bank and working cell banks can support the life cycle of an affordable, off-the-shelf NK cell product. We developed a robust stepwise protocol for large-scale generation of high-purity NK cells from human iPSCs without the cell isolation procedure. First, human iPSCs formed embryoid bodies and then were differentiated into highly purified (>98.0%) CD34 + CD43 + hematopoietic stem and progenitor cells (HSPCs) using a combination of cytokines and small molecles such as BMP-4, VEGF, BFGF, SCF, FLT-3 and ascorbic acid. During this period, we observed up to an 180-fold cell increase in the number of HSPCs compared with human iPSCs. Second, these HSPCs were differentiated into NK cells with FLT3, SCF, IL-3, IL-7, and IL-15 etc., with cell number increased approximately 9000-fold compared with iPSCs. In the final step, iPSC-derived NK cells were further expanded up to 8000-fold which enable us to produce 1E12 NK cells from less than 1 million iPSCs. After expansion, iNK cells became more homogenous and exhibited a more mature phenotype. More than 99.0% of cells expressed both CD45 and CD56, and more than 70% expressed CD16. The expression of activating and co-stimulatory receptors (NKG2D, NKp46,NKp44, NKp30, CD244 and CD226) of NK cells was also high in these iNK cells. Intrestingly, the cells exhibited robust anti-tumor efficacy against K562 (chronic myelogenous leukemia), RPMI 8226 (multiple myeloma). Furthermore, the cryopreserved iNK cells demonstrated dramatic cytotoxicity to U-87 MG cells (malignant gliomas) in 2D culture and the tumor organoid, indicating that iNK cells derived from our protocol can be used as off-the-shelf therapy for both hematological and solid tumors. We also observed that these iNK cells had variable cytotoxicity in other tumors. For example, our iNK were not obviously cytotoxic to the NCI-H929 cell line, derived from a plasmacytoma myeloma patient and expressing BCMA at a high level. To improve the specific cytotoxicity of NK cells to NCI-H929 cells, we transduced a anti-BCMA CAR to the donor iPSC. Following differentiation, the CAR-transduced iNK cells showed approximately 6-fold cytotoxicity compared with WT iNK. (Figure 1) Collectively, our serum-free and strom cell-free NK cell generation produre from human iPSCs provides a scalable, consistent, off-the-shelf and cost-effective NK/CAR-NK cell manufacturing platform for clinical application and NK cell-based immunotherapies.

Towards Clinical Translation of In Situ Cartilage Engineering Strategies: Optimizing the Critical Facets of a Cell-Laden Hydrogel Therapy

Background:Articular cartilage repair using implantable photocrosslinkable hydrogels laden with chondrogenic cells, represents a promising in situ cartilage engineering approach for surgical treatment. The development of a surgical procedure requires a minimal viable product optimized for the clinical scenario. In our previous work we demonstrated how gelatin based photocrosslinkable hydrogels in combination with infrapatellar derived stem cells allow the production of neocartilage in vitro. In this study, we aim to optimize the critical facets of the in situ cartilage engineering therapy: the cell source, the cell isolation methodology, the cell expansion protocol, the cell number, and the delivery approach.Methods:We evaluated the impact of the critical facets of the cell-laden hydrogel therapy in vitro to define an optimized protocol that was then used in a rabbit model of cartilage repair. We performed cells counting and immunophenotype analyses, chondrogenic potential evaluation via immunostaining and gene expression, extrusion test analysis of the photocrosslinkable hydrogel, and clinical assessment of cartilage repair using macroscopic and microscopic scores.Results:We identified the adipose derived stem cells as the most chondrogenic cells source within the knee joint. We then devised a minimally manipulated stem cell isolation procedure that allows a chondrogenic population to be obtained in only 85 minutes. We found that cell expansion prior to chondrogenesis can be reduced to 5 days after the isolation procedure. We characterized that at least 5 million of cells/ml is needed in the photocrosslinkable hydrogel to successfully trigger the production of neocartilage. The maximum repairable defect was calculated based on the correlation between the number of cells retrievable with the rapid isolation followed by 5-day non-passaged expansion phase, and the minimum chondrogenic concentration in photocrosslinkable hydrogel. We next optimized the delivery parameters of the cell-laden hydrogel therapy. Finally, using the optimized procedure for in situ tissue engineering, we scored superior cartilage repair when compared to the gold standard microfracture approach.CONCLUSION:This study demonstrates the possibility to repair a critical size articular cartilage defect by means of a surgical streamlined procedure with optimized conditions.

Quick and Easy Isolation of Immune Cells From Large-Volume Samples

Abstract Large-scale cell isolation is commonly performed in labs and core facilities for cell banking and drug discovery, and as a critical step in cell therapy manufacturing. Current methods can be a significant bottleneck in a lab’s workflow, often requiring a full day for sample processing and cell isolation. To address this need, we have developed two methods: manual column-free isolation using the Easy 250 EasySep™ magnet, and automated cell isolation in a closed system using RoboSep™-C. The Easy 250 magnet allows users to isolate cells from a full leukopak of up to 20 billion cells in under 30 minutes by simply pipetting out their target cells. Negative selection protocols have been optimized to achieve 95.3% T cell, 97.2% CD4+ T cell, 91.9% CD8+ T cell, 99.4% B cell, 96.5% NK cell, and 91.5% monocyte purities. Positive selection protocols obtain 95.4% CD3+ cell, 94.4% CD4+ T cell, 93.9% CD8+ T cell, and 96.2% CD14+ cell purities. RoboSep™-C automates this cell isolation procedure, along with the cell washing steps for sample preparation, in as little as 50 minutes. The instrument features a scale tower, pump and clamp modules to direct fluid through defined paths, and a magnet for separation of labelled cells. The system uses a sterile single-use tubing set that incorporates a cartridge for cell washing and concentration, and a magnet chamber for cell separation. Starting with fresh leukopaks, we obtained 95.9% T cell, 95.8% CD4+ T cell, and 89.6% CD8+ T cell purities following negative selection, and 92.0% CD4+ T cell and 89.5% CD8+ T cell purities following positive selection. These approaches offer efficient and user-friendly cell isolation that allow researchers to scale up their operations, and can be easily integrated upstream of existing workflows.

Urine-Derived Stem Cells for Regenerative Medicine: Basic Biology, Applications, and Challenges.

Regenerative medicine based on stem cell research has the potential to provide advanced health care for human beings. Recent studies demonstrate that stem cells in human urine can serve as an excellent source of graft cells for regenerative therapy, mainly due to simple, low-cost, and noninvasive cell isolation. These cells, termed human urine-derived stem cells (USCs), are highly expandable and can differentiate into various cell lineages. They share many biological properties with mesenchymal stem cells, such as potent paracrine effects and immunomodulation ability. The advantage of USCs has motivated researchers to explore their applications in regenerative medicine, including genitourinary regeneration, musculoskeletal repair, skin wound healing, and disease treatment. Although USCs have showed many positive outcomes in preclinical studies, and although the possible applications of USCs for animal therapy have been reported, many issues need to be addressed before clinical translation. This article provides a comprehensive review of USC biology and recent advances in their application for tissue regeneration. Challenges in the clinical translation of USC-based therapy are also discussed. Impact statement Recently, stem cells isolated from urine, referred to as urine-derived stem cells (USCs), have gained much interest in the field of regenerative medicine. Many advantages of human USCs have been found for cell-based therapy: (i) the cell isolation procedure is simple and low cost; (ii) they have remarkable proliferation ability, multidifferentiation potential, and paracrine effects; and (iii) they facilitate tissue regeneration in many animal models. With the hope to facilitate the development of USC-based therapy, we describe the current understanding of USC biology, summarize recent advances in their applications, and discuss future challenges in clinical translation.

Automated Large-Scale T Cell Isolation in a New Closed Cell Separation System

Abstract Large-scale T cell isolation is commonly performed in a wide range of laboratory settings, including for cell therapy and drug discovery research, as well as in core facilities as part of cell manufacturing and banking. However, current methods can be a significant bottleneck in a lab’s workflow, often requiring a full day just for sample processing and cell isolation. Furthermore, many applications have stringent requirements for purity, sterility, and standardization of the cell isolation procedure. To address these needs, we have developed RoboSep™-C, an instrument for efficient and automated cell isolation in a closed system. RoboSep™-C automates the established EasySep™ technology for immunomagnetic cell separation to enable isolation of untouched T cells, CD4+ T cells, or CD8+ T cells from leukapheresis samples. To set up the system, the user follows the on-screen prompts to install a sterile single-use tubing set and load the recommended medium, cell isolation reagents, and starting leukopak. The instrument then performs all necessary cell processing steps including sample washing, cell labeling, magnetic separation, and cell concentration in a 50-minute protocol. Starting with samples ranging from 2.5 to 20 billion nucleated cells, we obtained purities of 95.4 ± 4.1%, 95.7 ± 2.5%, and 89.3 ± 3.5% for T cell (n=16), CD4+ T cell (n=14), and CD8+ T cell (n=12) isolation, respectively (mean ± SD). The recoveries of the isolated T, CD4+ T, and CD8+ T cells were 64.2 ± 12.4%, 67.0 ± 8.2%, and 54.3 ± 13.5%, respectively. Automated isolation of high-purity T cells with RoboSep™-C enables researchers to scale up their operations and can be easily integrated upstream of existing T cell expansion, genome editing, and cryopreservation protocols.

Cell culture of differentiated human salivary epithelial cells in a serum-free and scalable suspension system: The salivary functional units model.

Saliva aids in digestion, lubrication, and protection of the oral cavity against dental caries and oropharyngeal infections. Reduced salivary secretion, below an adequate level to sustain normal oral functions, is unfortunately experienced by head and neck cancer patients treated with radiotherapy and by patients with Sjögren's syndrome. No disease-modifying therapies exist to date to address salivary gland hypofunction (xerostomia, dry mouth) because pharmacotherapies are limited by the need for residual secretory acinar cells, which are lost at the time of diagnosis, whereas novel platforms such as cell therapies are yet immature for clinical applications. Autologous salivary gland primary cells have clinical utility as personalized cell therapies, if they could be cultured to a therapeutically useful mass while maintaining their in vivo phenotype. Here, we devised a serum-free scalable suspension culture system that grows partially digested human salivary tissue filtrates composing of acinar and ductal cells attached to their native extracellular matrix components while retaining their 3D in vivo spatial organization; we have coined these salivary spheroids as salivary functional units (SFU). The proposed SFU culture system was sub-optimal, but we have found that the cells could still survive and grow into larger salivary spheroids through cell proliferation and aggregation for 5 to 10days within the oxygen diffusion rates in vitro. In summary, by using a less disruptive cell isolation procedure as the starting point for primary cell culture of human salivary epithelial cells, we demonstrated that aggregates of cells remained proliferative and continued to express acinar and ductal cell-specific markers.

Establishment of human trabecular meshwork cell cultures using nontransplantable corneoscleral rims.

Human trabecular meshwork (hTM) cell isolation in academic settings utilizes the motile nature of these cells, allowing them to migrate away from the explant and proliferate on distal regions of the culture substrate. Corneoscleral rims used for transplantation are a potential source of explants for the establishment of hTM cell cultures. However, cell isolation and the initiation of primary cell cultures from ocular tissues stored in Optisol-GS medium for an extended period of time (>6 days) has proven difficult, since Optisol-GS remarkably reduces cell viability and cellularity. Therefore, explants obtained from ocular tissues stored in Optisol-GS do not often provide adequate cell yield to initiate primary cell cultures if conventional culture techniques are used. Therefore, the majority of the research on primary hTM cell isolation has been accomplished using donor tissue obtained within 72 h postmortem. The goal of this study was to develop an hTM cell isolation procedure from nontransplantable ocular materials, utilizing the anchorage dependency of TM cells. This procedure yielded functionally viable cells, eficiently dissociated from the trabecular meshwork. Isolated cells demonstrated typical hTM cell characteristics including monolayer formation, contact inhibition, phagocytosis, and responses to glucocorticoid exposure. To the best of our knowledge, this is the first time an expired explant has been utilized in the successful isolation of hTM cells. Our results clearly demonstrate the advantage of increasing the anchor points of hTM cells for enhanced cell migration out from the explants, which have limited cell proliferative capacity.

Evidence of primary cilia in the developing rat heart

BackgroundA transient increase in cytosolic Ca2+ (the “Ca2+ transient”) determines the degree and duration of myocyte force development in the heart. However, we have previously observed that, under the same experimental conditions, the Ca2+ transients from isolated cardiac myocytes are reduced in amplitude in comparison to those from multicellular cardiac preparations. We therefore questioned whether the enzymatic cell isolation procedure might remove structures that modulate intracellular Ca2+ in some way. Primary cilia are found in a diverse range of cell types, and have an abundance of Ca2+-permeable membrane channels that result in Ca2+ influx when activated. Although primary cilia are reportedly ubiquitous, their presence and function in the heart remain controversial. If present, we hypothesized they might provide an additional Ca2+ entry pathway in multicellular cardiac tissue that was lost during cell isolation. The aim of our study was to look for evidence of primary cilia in isolated myocytes and ventricular tissue from rat hearts.MethodsImmunohistochemical techniques were used to identify primary cilia-specific proteins in isolated myocytes from adult rat hearts, and in tissue sections from embryonic, neonatal, young, and adult rat hearts. Either mouse anti-acetylated α-tubulin or rabbit polyclonal ARL13B antibodies were used, counterstained with Hoechst dye. Selected sections were also labelled with markers for other cell types found in the heart and for myocyte F-actin.ResultsNo evidence of primary cilia was found in either tissue sections or isolated myocytes from adult rat ventricles. However, primary cilia were present in tissue sections from embryonic, neonatal (P2) and young (P21 and P28) rat hearts.ConclusionThe lack of primary cilia in adult rat hearts rules out their contribution to myocyte Ca2+ homoeostasis by providing a Ca2+ entry pathway. However, evidence of primary cilia in tissue from embryonic and very young rat hearts suggests they have a role during development.

Isolation of Germ Cells From Testes of Stallions Using Collagenase and Trypsin-Ethylenediaminetetraacetic Acid

A germ cell isolation technique with two-enzyme digestion has been widely used to disaggregate testicular tissues for the preparation of individual germ cells. The main objective of this study was to assess the applicability of the two-enzyme germ cell isolation procedure to stallion testes. The testicular samples were obtained via field castration of prepubertal and postpubertal stallions. Then, equal amounts (10 g) of testicular tissue from each stallion were subjected to the two-enzyme germ cell isolation procedure with collagenase and trypsin-ethylenediaminetetraacetic acid. After the germ cells were isolated, viability and recovered germ cells were evaluated using trypan blue staining and a hemocytometer. Immunocytochemistry with UTF1 antibody was performed to evaluate the rate of undifferentiated spermatogonial stem cells. In addition, VASA antibody was used to measure the recovery rate for spermatogonia, spermatocyte, and round spermatid. The viability and recovery rate of the germ cells from prepubertal and postpubertal stallions were not statistically different. However, greater numbers of UTF1-positive germ cells (i.e., undifferentiated spermatogonia) were obtained from the testes of prepubertal stallions than from those of postpubertal stallions. In addition, the viability of germ cells isolated 2 days after castration did not differ from that of cells isolated on the same day that horses were castrated, and germ cells were successfully cryopreserved with 10% dimethyl sulfoxide for 1 month, although the viability was significantly reduced. In conclusion, the two-enzyme germ cell isolation procedure is applicable to the isolation of high-purity spermatogonia, spermatocyte, or round spermatid from stallion testes.

The first steps towards generating induced pluripotent stem cells from cryopreserved skin biopsies of marine mammals

A new approach for restoring populations of endangered marine mammal species is to use induced pluripotent stem cells (iPSCs). These cells can be obtained by genetic reprogramming of somatic animal cells, such as skin fibroblasts. From there, the iPSCs can be differentiated into all cell types of the organism, including spermatozoa and oocytes. The skin biopsies of three species of marine mammals, belonging to different orders (the walrus Odobenus rosmarus, the Steller sea lion Eumetopias jubatus and the Irrawaddy dolphin Orcaella brevirostris), were taken and cryopreserved. The fibroblast cultures were obtained from thawed skin biopsies of sea lion and walrus using previously developed methods for human cells. In contrast, Irrawaddy dolphin skin cells did not survive this cell isolation procedure. We had to modify the isolation method to obtain a fibroblast culture from this animal, using mechanical disaggregation only and a higher concentration of serum and antibiotics at the first steps of cultivation. The functionally active fibroblasts will be used in the next step, generating iPSCs. Testing the quality of the fibroblast cultures, we found that the abnormal fibroblasts differed in size and the distribution of filamentous actin and tubulin, compared to normal cells, and demonstrated clear aberrations in the nuclei.

Optimization of EGFR high positive cell isolation procedure by design of experiments methodology.

Circulating tumor cells (CTCs) in blood circulation may play a role in monitoring and even in early detection of metastasis patients. Due to the limited presence of CTCs in blood circulation, viable CTCs isolation technology must supply a very high recovery rate. Here, we implement design of experiments (DOE) methodology in order to optimize the Bio-Ferrography (BF) immunomagnetic isolation (IMI) procedure for the EGFR high positive CTCs application. All consequent DOE phases such as screening design, optimization experiments and validation experiments were used. A significant recovery rate of more than 95% was achieved while isolating 100 EGFR high positive CTCs from 1 mL human whole blood. The recovery achievement in this research positions BF technology as one of the most efficient IMI technologies, which is ready to be challenged with patients' blood samples.

A novel enzyme blend for efficient tissue dissociation and primary cells isolation

Tissue dissociation/primary cell isolation and cell harvesting are principal appli- cations for enzymes in tissue culture research and cell biology studies. The goal of a cell isolation procedure is to maximize the yield of functionally viable dissoci- ated cells. Among the parameters which affect the outcome of any particular dissociating procedure there are enzyme(s) used and related impurities presents in crude enzyme preparation. ABIEL srl recently produced the recombinant collagenase class I (Col G) and II (Col H) from Clostridium histolyticum (PCT WO 2011/073925 A9). The enzymes were produced in Escherichia coli and purified by affinity chromatography. The method of production adopted allows absolute control of the final composition of these enzymes, as well as their stability, purity, activity, absence of toxicity and higher reproducibility of batches. The two collagenases produced separately have been used in conjunction according to precise proportions to dissociate calvaria, liver, pancreas, retina of the BALB/c mouse; and bovine hoof. The analyses carried out on all isolated cell populations suggest that the cells maintain the structural and functional integrity of specific tissues/organs originating. Recombinant Col G and Col H enzymes produced by ABIEL are promising in the context of the tissue/cells dissociation, with the aim to make innovation in the fields of tissue engineering and transplantation medicine.

Folic acid functionalized surface highlights 5-methylcytosine-genomic content within circulating tumor cells.

Although the detection of methylated cell free DNA represents one of the most promising approaches for relapse risk assessment in cancer patients, the low concentration of cell-free circulating DNA constitutes the biggest obstacle in the development of DNA methylation-based biomarkers from blood. This paper describes a method for the measurement of genomic methylation content directly on circulating tumor cells (CTC), which could be used to deceive the aforementioned problem. Since CTC are disease related blood-based biomarkers, they result essential to monitor tumor's stadiation, therapy, and early relapsing lesions. Within surface's bio-functionalization and cell's isolation procedure standardization, the presented approach reveals a singular ability to detect high 5-methylcytosine CTC-subset content in the whole CTC compound, by choosing folic acid (FA) as transducer molecule. Sensitivity and specificity, calculated for FA functionalized surface (FA-surface), result respectively on about 83% and 60%. FA-surface, allowing the detection and characterization of early metastatic dissemination, provides a unique advance in the comprehension of tumors progression and dissemination confirming the presence of CTC and its association with high risk of relapse. This functionalized surface identifying and quantifying high 5-methylcytosine CTC-subset content into the patient's blood lead significant progress in cancer risk assessment, also providing a novel therapeutic strategy.

On the Path to Autologous IVD Regeneration?

Introduction Intervertebral disc (IVD) degeneration is often the cause of low back pain. With age, extracellular matrix (ECM) depletion occurs, leading to nucleus pulpous (NP) extrusion and IVD destruction. Concomitantly, there is a decline in viable cell populations, which in turn remain poorly characterized. As current clinical treatments have not provided adequate solutions, we propose an in-depth study in terms of NP cell subpopulation characterization and ECM changes with aging, in view of regeneration. Materials and Methods The first step was to characterize NP cell populations harvested from bovine IVDs, to better understand IVD's potential to endogenously regenerate. We started by optimizing cell isolation procedure, still not consensual among the literature. For that, distinct digestive enzymes (collagenase type-I, collagenase type-II, and collagenase type-XI) were used at three different concentrations (0.5, 1.0, and 2.0 mg/mL, respectively), for 4 and 19 hours. To understand the cellular effect of the digestion, we evaluated the cell yield (trypan blue exclusion); cell viability/apoptosis (AnnexinV/Propidium Iodide staining, Flow Cytometry), and cell morphology (actin (phalloidin-AlexaFluor488) and nuclei (DRAQ-5) staining, Imaging Flow Cytometry). The presence of specific cell subpopulations within NP and its phenotype was then investigated, by assessing the expression of CD29, CD44, CD45, CD146, CD34, Gly-A, and Brachyury. Due to microenvironments’ growing importance for regeneration, in parallel, we compared the NP ECM proteomic profile of bovine IVDs from fetus, young and old animals by quantitative iTRAQ LC-MS/MS. The candidates differentially expressed in the three age groups are being validated by Western blot. Results Cell yield and viability improved, in a concentration dependent manner, with increasing collagenase activity against collagen I and II—collagenase XI rendered the highest cell yield and greatest viability. In addition, cell yield was higher for shorter digestion periods (4 hours). Furthermore, NP cells did not reveal major morphological changes dependent on the enzymes used. Interestingly, three viable subpopulations, with different sizes and autofluorescence, were consistently identified by flow cytometry, immediately after isolation. Expression of cell markers differed among these subpopulations, proving the existence of an heterogeneous cell population within the NP. Concerning iTRAQ analysis, a few interesting candidates emerged that are expressed in different proportions in fetus, young and old animals. These quantitative differences are being validated. Conclusion This study shows that collagenase type XI is the most efficient enzyme to be used for isolation and that different cell populations can be identified within the bovine NP. It also demonstrates that distinct ECM proteins are expressed in the IVD at different ratios depending on bovine age groups. Acknowledgments This work was financed by FEDER funds through the Programa Operacional Factores de Competitividade—COMPETE, by Portuguese funds through FCT— Fundação para a Ciência e a Tecnologia. The authors would also like to acknowledge the AO SPINE Research Network (AO SRN) for funding. Disclosure of Interest None declared

115-P: AUTOMATED HIGH THROUGHPUT ISOLATION OF LYMPHOID AND MYELOID CELLS DIRECTLY FROM WHOLE BLOOD USING RoboSep-HT

Aim Process automation is an important requirement for streamlining and standardizing technical procedures, such as cell isolation, in a routine laboratory. We have previously developed RoboSep™, the first instrument to fully automate the cell labeling and separation steps of an immunomagnetic cell isolation procedure. RoboSep™ uses EasySep™ reagents and protocols and can isolate up to 4 individual samples at one time. More recently we have developed a high throughput version of RoboSep™, called RoboSep-HT, which can isolate 16 samples from up to 4 different cell types in about 1 hour. Like RoboSep™, RoboSep-HT uses disposable filter tips to avoid cross contamination between samples and is operated using an intuitive graphical user interface. Sample barcoding is a standard feature. In this study, positive selection of CD3+ and myeloid+ cells directly from whole blood using EasySep™ reagents was performed on the new RoboSep-HT and compared to performance using the classic RoboSep™. Methods 1mL of fresh, 24-hour or 48-hour old whole blood was diluted with an equal volume of 1xRBC lysis buffer and loaded on each instrument. Purity (flow cytometry), recovery (cell counts) and DNA yield (OD) were assessed following isolation using each robot. Replicates were averaged prior to statistical analysis (t-tests). Results There was no significant difference in either purities, cell recoveries or DNA yields when CD3 and myeloid cell isolation was performed on the RoboSep-HT versus the classic RoboSep™. [ Table 1 ] Conclusions RoboSep-HT can be used for the high throughput isolation of multiple cell types and is ideally suited for cell isolation prior to chimerism testing. McQueen: STEMCELL Technologies: Employee. Kellerman: STEMCELL Technologies: Employee. Frehlick: STEMCELL Technologies: Employee. Gromadzki: STEMCELL Technologies: Employee. Poon: STEMCELL Technologies: Employee. Woodside: STEMCELL Technologies: Employee. Thomas: STEMCELL Technologies: Employee.

Nrf2 signalling promotes ex vivo tubular epithelial cell survival and regeneration via murine double minute (MDM)-2

Tubular repair upon injury involves regeneration from either surviving tubular epithelial cells or from their surviving local progenitor cells; hence, compound screening with cell lines may be inadequate. Here, we demonstrate that the renal cell isolation procedure and subsequent outgrowth of tubular cells can mimic the renal injury phase and tubular cell regeneration from whichever surviving renal cells. We set up assays to systematically screen and identify mediators of tubular survival and repair. Forty-eight hours after plating total kidney isolates from C57BL/6 mice, 69% of cells survived when prepared from 2-week-old pups, but only 4% of cells from 8-week-old mice, respectively. This poor survival was not modulated by co-incubation with any of 24 cytokines and growth factors, except for the Nrf2 agonist sulforaphane. In addition, only sulforaphane enhanced the regenerative outgrowth of tubular epithelial cells from the mixed population. Furthermore, sulforaphane enhanced wound closure upon scratching tubular epithelial cell monolayers in a dose-dependent manner. This process was associated with the induction of the tested Nrf2 target genes HO-1, NQO1 and murine-double minute 2 (MDM2). MDM2 blockade with nutlin-3a completely blocked the protective effects of sulforaphane on renal cell survival, outgrowth and wound closure. Together, renal cell isolation is a model of acute kidney injury (AKI). Primary tubular epithelial cell outgrowth represents a model of tubular regeneration. Nrf2 activation can enhance renal cell survival and tubular repair by inducing the cell cycle regulator MDM2.

Isolation of multipotent neural stem or progenitor cells from both the dentate gyrus and subventricular zone of a single adult mouse

In adult mammals, the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone of the dentate gyrus (DG) show ongoing neurogenesis, and multipotent neural stem or progenitor cells (NSCs) in these two regions exhibit different intrinsic properties. However, investigation of the mechanisms underlying such differences has been limited by a lack of efficient methods for isolating NSCs, particularly from the adult DG. Here we describe a protocol that enables us to isolate self-renewing and multipotent NSCs from the SVZ and the DG of the same adult mouse. The protocol involves the microdissection of the SVZ and DG from one adult mouse brain, isolation of NSCs from specific regions and cultivation of NSCs in vitro. The entire procedure takes 2-3 h. As only one mouse is needed for each cell isolation procedure, this protocol will be particularly useful for studies with limited availability of mice, such as mice that contain multiple genetic modifications.

Differences in transcriptional patterns of extracellular matrix, inflammatory, and myogenic regulatory genes in myofibroblasts, fibroblasts, and muscle precursor cells isolated from old male rat skeletal muscle using a novel cell isolation procedure

Aged skeletal muscle displays increased fibrosis and impaired regeneration. While it is not well characterized how skeletal muscle fibroblasts contribute to these phenomena, transforming growth factor-β1 (TGF-β1) and Delta/Notch signaling have been implicated to influence muscle regeneration. In this study, a unique combination of aging phenotypes is identified in differentiating fibroblasts (myofibroblasts), proliferating fibroblasts, and muscle precursor cells (MPCs) that characterize an impaired regenerative potential observed in aged skeletal muscle. Using a novel dual-isolation technique, that isolates fibroblasts and MPCs from the same rat skeletal muscle sample, and cell culture conditions of 5 % O(2) and 5 % CO(2), we report for the first time that myofibroblasts from 32-mo-old skeletal muscle, compared to 3-mo-old, display increased levels of mRNA for the essential extracellular matrix (ECM) genes, collagen 4α1 (83 % increase), collagen 4α2 (98 % increase), and laminin 2 (113 % increase), as well as increased levels of mRNA for the inflammatory markers, interleukin-6 (4.3-fold increase) and tumor necrosis factor α (3.2-fold increase), and TGF-β1 (84 % increase), whose protein controls proliferation and differentiation. Additionally, we demonstrate that proliferating fibroblasts from 32-mo-old skeletal muscle display increased levels of mRNA for the Notch ligand, Delta 1 (≥2.0-fold increase). Together, these findings suggest that increased expression of ECM and inflammatory genes in myofibroblasts from 32-mo-old skeletal muscle may contribute to the fibrogenic phenotype that impairs regeneration in aged skeletal muscle. Furthermore, we believe the novel dual-isolation technique developed here may be useful in studies that investigate communications among MPCs, fibroblasts, and myofibroblasts in skeletal muscle.