The study of immune cell function in non-lymphoid tissue and tumors promises to elucidate novel strategies to treat immune disorders, infectious diseases, and cancer. To address the challenge of isolating leukocytes from complex and variable tissues and tumors, we have developed a new protocol to isolate particle-free, human CD45+ leukocytes. Using the EasySep™ Release Human CD45 Positive Selection Kit, leukocytes are labeled with antibody complexes linked to magnetic particles and separated using an EasySep™ magnet. The magnetic particles are then removed from the desired cells by resuspension in EasySep™ Release Buffer and a final magnetic separation. To assess performance, NRG-3GS mice were first engrafted with human CD34+ cells followed by xenotransplant with human breast (MDA-MB-231) or ovarian (SKOV3) cancer cell lines. In humanized mouse lungs, bone marrow and spleen, the starting and isolated human CD45+ frequency ranges were 6.0 – 57.2% and 90.9 – 99.4%, respectively (n = 3). Starting with human tumor xenografts, tumor infiltrating leukocytes were enriched from a starting range of 0.4 – 18.0% to 76.6 – 92.7% (n = 4). The final immune cell frequencies are representative of the starting population, and further separation of immune subsets can be achieved with additional downstream isolation. Humanized mouse models of clinical disease are instrumental in furthering our understanding of complex mechanisms of disease progression and resolution. This new kit for the isolation of human immune cells from tissues and tumors will facilitate further examination of the roles of immunity in disease and the evaluation of immune-based treatment strategies.

The increasing use of high dose ablative chemo/radiation therapy followed by hematopoietic cell rescue challenges our knowledge of the biology of the cells responsible for reconstitution of the hematopoietic system and of the factors contributing to disease relapse. Purification of stem cells via negative selection involves antibody labeling and removal of a vast majority of the cells in the start suspension. This may require more antibody/reagents and the ability to bind a larger number of cells. Purification of stem cells via negative selection involves antibody labeling and removal of a vast majority of the cells in start suspension. This may require more antibody/reagents and the ability to bind a larger number of cells. Patients have been successfully transplanted with purified cell suspensions where the progenitors and stem cells have been previously labeled with antibodies. In summary, technical advances in scale up and cell labeling have produced a large scale lineage depletion technique capable of purifying human stem/progenitor cells with efficiencies.

Organoids are stem cell-derived structures that are generated in three-dimensional tissue culture. They are unique since they exhibit a high degree of self-organization and differentiation, and thus recapitulate many of the features of the tissues from which they were derived. Because of this, organoids are now firmly established as an essential tool in medical research, and have the potential to drastically reduce the number of animals required for experimentation.

Studies of hematopoietic stem and progenitor cells (HSPCs) in Acute and Chronic Myeloid Leukemia (AML and CML) are often hampered by limited access to patient samples and poor yield/viability of cryopreserved cells. We developed a culture system that enables efficient expansion of HSPCs from most CML and AML patients. CD34+ cells were isolated from previously frozen AML and CML samples (n=6 each) and cultured (104 cells/mL) in serum-free StemSpan™ SFEM II medium with CD34+ Expansion Supplement (containing SCF, TPO, FLT3L, IL-3, IL-6). After 7 and 14 days, the number, immunophenotype, aldehyde dehydrogenase (ALDH) activity and colony-forming potential of the expanded cells were measured. Culture of CD34+ cells on a pre-established monolayer of normal marrow-derived mesenchymal stromal cells (MSCs) improved output of CD34+, CD34+CD90+CD45RA- and ALDH+ cells and CFUs by >2-fold vs. stroma-free cultures. Addition of the small molecule UM171 also increased cell outputs and effectively replaced the need for MSCs. The frequency of CD34+ cells after 7 and 14 days was 80% and 50% with and 40% and 15% without UM171, respectively. CD34+ cell numbers increased ∼65-fold (CML) and ∼30-fold (AML) after 7 days with UM171 and were ∼10-fold (CML) and ∼3-fold (AML) higher than without UM171 (p

Background & Aim MesenCult™-ACF-Plus (MACF-P) is an improved animal component-free (ACF) culture medium for the derivation and expansion of mesenchymal stromal cells (MSCs) from primary tissues. We characterized MSCs derived from human bone marrow (BM) and adipose (AD) tissues in MACF-P and in medium containing fetal bovine serum (FBS). Methods, Results & Conclusion Clonogenic growth was evaluated by plating BM mononuclear cells (MNCs) or AD-derived stromal cells (SCs) at low density in the Colony-Forming Unit-Fibroblast (CFU-F) assay. The proliferative potential of BM and AD-derived MSCs was measured by determining cell number at each passage (P) up to P6 (BM-MSCs) or P9 (AD-MSCs), and paired t-test was used for statistical analysis. MSCs from both tissue sources were plated at 1.5–3 × 103 cells/cm2 in each medium for long-term cell expansion. Immunosuppression of CD4+ T cells by BM-MSCs cultured in MACF-P was evaluated by co-culture of BM-MSCs with PBMCs. The PBMCs were labelled with eFluor450, activated with human CD3/CD28 T Cell Activator and then analyzed by flow cytometry for T cell proliferation after 5 days. Total CFU-F per 106 AD-SCs and per 106 BM-MNCs was comparable in MACF-P and FBS containing medium (AD-SCs: 236 ± 127 vs. 268 ± 120 [mean ± SEM; n=3]; BM-MNCs: 37 ± 7 vs. 32 ± 5 [mean ± SEM; n=6] in MACF-P and FBS-containing media, respectively). However, the average fold-expansion of AD-MSCs at each subculture over 9 passages was significantly higher in MACF-P medium (12.2 ± 0.9; mean ± SEM; n=5) than in FBS-containing medium (3.8 ± 0.3; mean ± SEM; n=5; p<0.05). Similarly, the average fold-expansion of BM-MSCs at each subculture over 6 passages was also significantly higher in MACF-P (11.6 ± 1.2; mean ± SEM; n=5) than in FBS-containing medium (3.9 ± 0.4; mean ± SEM; n=5; p<0.05). Both AD-MSCs and BM-MSCs cultured in MACF-P differentiated more robustly in vitro under the appropriate conditions into adipogenic, osteogenic and chondrogenic cells (as visualized by Oil Red O, Alizarin Red and Alcian Blue staining, respectively) compared to the same cells cultured in FBS-containing medium. Co-culture of BM-MSCs with PBMCs in MACF-P indicated that proliferation of CD4+ T cells is suppressed in a cell concentration-dependent manner when BM-MSCs were added in vitro. These data demonstrate that improved MACF-P medium supports derivation and efficient expansion of both BM-MSCs and AD-MSCs under complete ACF conditions and that BM-MSCs exhibit immunosuppressive activity in vitro. MesenCult™-ACF-Plus (MACF-P) is an improved animal component-free (ACF) culture medium for the derivation and expansion of mesenchymal stromal cells (MSCs) from primary tissues. We characterized MSCs derived from human bone marrow (BM) and adipose (AD) tissues in MACF-P and in medium containing fetal bovine serum (FBS). Clonogenic growth was evaluated by plating BM mononuclear cells (MNCs) or AD-derived stromal cells (SCs) at low density in the Colony-Forming Unit-Fibroblast (CFU-F) assay. The proliferative potential of BM and AD-derived MSCs was measured by determining cell number at each passage (P) up to P6 (BM-MSCs) or P9 (AD-MSCs), and paired t-test was used for statistical analysis. MSCs from both tissue sources were plated at 1.5–3 × 103 cells/cm2 in each medium for long-term cell expansion. Immunosuppression of CD4+ T cells by BM-MSCs cultured in MACF-P was evaluated by co-culture of BM-MSCs with PBMCs. The PBMCs were labelled with eFluor450, activated with human CD3/CD28 T Cell Activator and then analyzed by flow cytometry for T cell proliferation after 5 days.

The study of immune cell function in non-lymphoid tissue and tumors has emerged as an exciting research area, with the promise to provide novel strategies for the treatment of immune disorders, infectious diseases and cancer. A major challenge in the field is the isolation of leukocytes from tissues due to the complexity of starting samples that can be highly variable in frequency and subset composition. We describe a new 28 minute protocol optimized for the isolation of particle-free human CD45+ leukocytes from varied sample sources. Using the EasySep™ Release Human CD45 Positive Selection Kit, leukocytes are labeled with antibody complexes linked to magnetic particles and separated using an EasySep™ magnet. The isolated cells are then released from particles by resuspension in EasySep™ Release Buffer and a final magnetic separation step. Performance was assessed using human leukocytes spiked into a single cell suspension of mouse thymocytes across a range of frequencies to simulate the variable leukocyte content in tissue and tumor samples. Human CD45+ purities were 95.4 ± 4.3% from start ranges of 0.5–2.0% (n=15) and 99.1 ± 0.7% from start frequencies of ≥10% (n=18). The isolated cells are compatible with further downstream isolation and are functional, as demonstrated by T cell proliferation and IFN-gamma production assays. We have also assessed our CD45+ isolation kit from tissues of humanized NSG mice, expanding the application of our protocol to highly relevant disease and cancer models. The EasySep™ Release Human CD45 Positive Selection Kit provides a robust method for high purity isolation from diverse tissues and starting frequencies, generating particle-free cells ready for downstream evaluation.

Immune cell function is often tissue-specific, therefore isolating cells from their tissue microenvironment is necessary to better understand their role in health and disease. This can be challenging in the presence of non-immune, tissue-derived cells and cellular debris from tissue dissociation. These factors reduce the leukocyte start frequency, which can prolong the isolation process and limit the identification of small but critical leukocyte subsets. To overcome these obstacles, we developed a simple and rapid selection method to enrich for CD45+ leukocytes from mouse tissues. Starting with a single cell suspension, cells are labelled with an antibody complex that links CD45+ cells to magnetic particles and following magnetic separation, the isolated CD45+ cells are ready for use. Using healthy mouse lung tissue as an example, CD45+ leukocytes were enriched from 63.5 ± 9.4% to 97.1 ± 1.2% purity, and the recovery of viable CD45+ cells was 37.7 ± 14.5% (mean ± SD, n=37). Preliminary testing on tumors from a mouse 4T1 breast tumor model resulted in efficient enrichment of tumor-infiltrating leukocytes. Importantly, the composition of immune subsets from both healthy lung tissues and tumor samples was maintained following CD45 selection. To demonstrate functionality, T cells within the CD45 selected population from spleen were able to upregulate CD25 and CD69, and proliferate upon stimulation. In as little as 20 minutes, the EasySep™ Mouse CD45 Isolation Kit allows researchers to easily enrich for functional leukocytes from tissue samples. Examining immune cells directly from healthy and tumor tissues will enhance our understanding of how these cells function and aid in developing new approaches to combat disease progression.

Erythroid cells generated from human pluripotent stem cells (hPSCs) can potentially offer an unlimited and safe supply of red blood cells (RBCs) for transfusion. Human PSC-derived erythroid cells at various stages of differentiation can also be used to model blood diseases, test new drug candidates, and develop cellular and genetic therapies. Although several protocols for deriving RBCs from hPSCs have been described, these are typically complex, involving multiple culture steps that may include co-culture with feeder cells, and exhibit large variability in erythroid cell yields between hPSC lines and replicate experiments. We have developed a straightforward, serum-free and feeder-free culture method to generate erythroid cells from hPSCs with high yields and high purity. The method has been validated on multiple human embryonic stem (ES) cell lines (H1, H7, H9) and induced pluripotent stem (iPS) cell lines (WLS-1C, STiPS-F016, STiPS-B004). The protocol involves two steps: hematopoietic specification of hPSCs, followed by differentiation of hPSC-derived hematopoietic stem and progenitor cells (HSPCs) into erythroid cells. Lineage specification and differentiation is driven by only three supplements that combine cytokines and other factors to support optimal differentiation efficiency and cell yield across cell lines. In the first step, small hPSC aggregates routinely maintained in feeder-free maintenance medium, are plated onto matrigel-coated microwells, and specification to mesoderm and subsequent hematoendothelial differentiation is induced by addition of successive expansion supplements. This phase promotes extensive hematopoietic progenitor cell generation, with a single hPSC producing on average 142 HSPCs (range: 50 - 360, n = 3 experiments) by day 10 across all six ES and iPS cell lines tested. The average frequency of cells expressing CD43, an embryonic pan-hematopoietic marker, is 92% (range: 85 - 95%), and the frequency of CD34+ cells ranges between 24-55%. In the second, erythroid differentiation step, hPSC-derived HSPCs expand on average 300-fold (range: 80 - 1000) within 10 - 14 days, and the average frequency of GlyA+ cells is 75% (range: 70 - 85%). Cumulatively, this results in the generation of on average 30,000 GlyA+ cells (range: 10,000 - 80,000) per initial hPSC after 20 - 24 days. Further maturation in 7-day cultures containing EPO and human serum resulted in a > 90% pure population of GlyA+ erythroid cells. Notably, no cell loss was observed during the maturation phase, resulting in an average yield of 50,000 GlyA+ cells (range: 10,000 - 220,000) per single initial hPSC on day 31. Differentiated cells were characterized by orthochromatic normoblast morphology and decreased CD71 expression, consistent with erythroid maturation. Erythroid cells generated in this differentiation culture system expressed a mix of 'primitive' and 'definitive' hemoglobin types, but with adult and fetal hemoglobin being expressed at higher levels than embryonic hemoglobin. The observed enucleation rates of hPSC-derived erythroid cells are consistent with current reports and are subject to further optimization. In summary, we have developed a two-step, serum- and feeder-free erythroid differentiation method to generate large numbers of erythroid cells from multiple hPSC lines. This culture system provides a simple, standardized and reproducible platform to generate RBCs from hPSCs with high yields and efficiency for basic and translational research. Disclosures Walasek: STEMCELL Technologies, Inc: Employment. Chau:STEMCELL Technologies, Inc: Employment. Barborini:STEMCELL Technologies, Inc: Employment. Richardson:STEMCELL Technologies, Inc: Employment. Szilvassy:STEMCELL Technologies, Inc: Employment. Louis:STEMCELL Technologies, Inc: Employment. Thomas:STEMCELL Technologies, Inc: Employment. Eaves:STEMCELL Technologies, Inc: Employment. Wognum:STEMCELL Technologies, Inc: Employment.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that regulate immune responses in cancer, chronic infections and inflammatory conditions. In an individual with a tumor burden, MDSCs accumulate in peripheral lymphoid organs and within the tumor microenvironment. In several human cancers, and in mouse tumor models, the presence of high numbers of MDSCs correlates with tumor growth, metastasis and an overall poor prognosis. Approaches that impair the activity or deplete MDSCs have shown promise in animal models. Thus, MDSCs are an attractive target for therapeutic intervention. We have developed an immunomagnetic method for the isolation of untouched mouse MDSCs. Using CyTOF analysis of cell surface antigens on spleen cells from tumor bearing mice, we developed a cocktail of antibodies that targets non-MDSCs for removal using a column-free, negative selection protocol. Our method isolates CD45+CD11b+Gr1+ cells with the following purity and recovery from spleen, BM and blood: MDSCs (CD11b+Gr1+) from tumor-bearing miceTissuePurity (±SD)Recovery (±SD)Spleen94 ± 2.1%50 ± 12%Blood99 ± 0.6%55 ± 7.0%Bone marrow96 ± 1.0%68 ± 5.6% (naïve) As a functional test, naïve mouse splenocytes were labeled with a proliferation dye and the T cells were activated with anti-CD3 and anti-CD28 antibodies. MDSCs isolated from tumor-bearing mice were added at different ratios and after 3 days of culture proliferation of CD4+ and CD8+ T cells was assessed by flow cytometry. This assay showed a dose-dependent suppression of both CD4+ and CD8+ T cells by the isolated MDSCs. Our new EasySep™ Mouse MDSC (CD11b+Gr1+) Isolation Kit offers a fast and easy method that could facilitate MDSC discoveries that impact a wide range of diseases.

Cancer immunotherapy using CAR T-cells is a rapidly progressing field and manufacturing these cells is a complex process that requires multiple optimization steps. We have developed reagents for the isolation, activation and expansion of human T cells that will be available for clinical cell therapy manufacturing. Soluble ImmunoCult™ Human T Cell Activators induce T cell activation via cross-linking CD3 and co-stimulatory molecules on the surface of cells. Activated T cells then can be genetically modified and subsequently expanded in ImmunoCult™-XF, a serum- and xeno-free T cell expansion medium. Here, we present several optimization strategies with ImmunoCult™ products in order to obtain high transfection efficiency and maximum cell yield. By evaluating activation dynamics of T cells and determining the optimal transfection time points, the transfection efficiency can be substantially improved in both CRISPR/Cas9- and lentiviral-mediated gene-modification methods. Our study also suggests that maintaining T cells at lower cell density after the third day following activation greatly improves cell viability and cumulative cell growth, resulting in an >1000-fold expansion of total human T cells with >85% viability over 10–12 days of culture. Expanded T cells co-express CD45RO+CD62L+ with low expression of PD-1. As an example, we applied the workflow described here to generate TCRαβ KO T cells from healthy donors with up to 90% knockout efficiency. The purity of TCRαβ KO T cells can be further increased with the use of an EasySep™ Human TCRαβ depletion kit. Taken together, the processes outlined in this study can be easily and rapidly implemented to improve T cell manufacturing efficacy.

Innate lymphoid cells (ILCs) are exceedingly rare but important regulators of homeostatic and disease-associated immune processes. The frequency of ILCs in peripheral blood of healthy humans is ~0.07% of CD45+ leukocytes. ILCs lack specific cell surface markers but can be divided into distinct subsets (ILC1, 2 and 3) based on their differential expression of effector cytokines and master transcription factors. Currently, cell sorting is the most widely used method to isolate ILCs, but it is time consuming, expensive and often results in low purities and recoveries. Pre-enrichment of ILCs would allow for reduced sorting times and improved purities. Accordingly, we have developed a fast immunomagnetic negative selection method to pre-enrich all ILCs subsets from human leukapheresis samples. Briefly, unwanted cells are labelled with antibodies and magnetic particles and placed into an EasySep™ magnet. Unwanted cells are retained in the magnet and the enriched ILC fraction is simply poured off into a new tube. We find that total ILCs (defined as Lineage− CD45+ CD127+) are enriched from a frequency of 0.01 – 0.23% (n=28) to a final frequency of 17 – 86%, an enrichment of 200 – 1500 fold with virtually no loss of ILCs. ILC1 were enriched from 0.01 – 0.2% to 4.5 – 14%. ILC2 were enriched from 0.01 – 0.1% to 5.8 – 51% and ILC3 were enriched from 0.01 – 0.1% to 6 – 16%. This pre-enrichment drastically decreases sort time, allowing sorting over 3.7 × 105 ILCs from 2 × 109 PBMCs in only 12 minutes. Sorted cells maintained their functionality; when stimulated, ILC1s produced IFNγ, ILC2s secreted IL-13 and ILC3s produced IL-22. Our newly developed method of ILC pre-enrichment should aid human ILC research by enabling their rapid isolation when combined with cell sorting

Peripheral blood circulating T cells can be subdivided into either antigen experienced effector and memory T cells or naïve T cells that have not yet encountered their cognate antigen. Naïve T cells are valuable tools when studying mechanisms of T cell activation and are required for foundational research into infectious diseases, cancer and transplantation. We describe here a simple immunomagnetic cell isolation protocol for the isolation of untouched human naïve T cells from fresh or previously frozen PBMC or leukapheresis samples. The column-free, negative selection procedure involves labelling and removing unwanted cells using bispecific antibody complexes that crosslink cell surface antigens to magnetic particles. The labelling cocktail is composed of antibodies that target non-T cells, memory T cells, and an optional cocktail for depleting gamma delta T cells. Briefly, the procedure involves a five minute incubation with the antibody cocktail followed by the addition of magnetic particles and two three minute magnetic separations in a hand-held EasySep™ magnet. Following the second magnetic separation, the untouched naïve T cells are simply poured off into a new tube and are ready for use. We define naïve T cells phenotypically as CD3+CD45RA+CD45RO-CD197+. Starting from fresh PBMC, we were able to obtain purities of 96.1 ± 2.3% (mean ± SD, n=14) and recoveries of 62.6% ± 24.5% (mean ± SD, n=14). Additionally, isolated naïve T cells are functional and respond by upregulating the expression of CD25 and CD69 upon stimulation. Our EasySep™ Human Naïve Pan T Cell Isolation Kit offers a fast and easy isolation method for naïve T cells that is suitable for downstream applications such as flow cytometry, cell culture or DNA/RNA extraction.

Growing prostate epithelial cells as organoids in a three-dimensional (3D) cell culture environment represents a more physiological model system than conventional 2D adherent cell culture systems for studying many different aspects of prostate epithelial cell biology. Two types of prostate epithelial organoids have been previously described: large hollow cystic organoids, and smaller solid organoids (Karthaus et al, Cell 2014). Both types of organoids are composed of androgen receptor (AR)-expressing luminal cells and keratin (K) 5-expressing basal cells, but only vary in the proportion of each cell type, with the cystic organoids enriched for luminal cells and solid organoids enriched for basal cells. We are currently developing ProstaCult™ Organoid Growth Medium, a serum-free medium for the long-term propagation of both types of organoids from mouse prostate tissue. To initiate the cultures, mouse prostates are enzymatically dissociated sequentially in collagenase type II, trypsin and dispase to generate a single cell suspension, and 5x103 the liberated cells are then embedded in Corning® Matrigel® and cultured in ProstaCult™ Organoid Growth Medium. Approximately 10±2% (mean±sem; n=6) of the seeded cells will proliferate and generate approximately equivalent numbers of cystic and solid organoids, which can be dissociated and passaged every 7 days as a single cell suspension. Typical total epithelial cell expansion observed during media development ranges from 21-240-fold per passage for a minimum of 4 passages. Experiments are ongoing to determine the long-term passageability of organoids maintained in ProstaCult™ Organoid Growth Medium. Organoids that are generated at multiple passages are composed of a polarized epithelium with AR+K18+ luminal cells and K5+ basal cells. These results demonstrate that ProstaCult™ Organoid Growth Medium efficiently generates and expands prostate epithelial organoids. Citation Format: John Stingl, David Rowbotham, Terry E. Thomas, Allen C. Eaves, Sharon A. Louis. Expansion of mouse prostate epithelial stem cells in serum-free ProstaCult Organoid Growth Medium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3111.

Tissue specific research often requires mechanical and/or enzymatic digestion to isolate and study certain cell types. The digestion can be a harsh process resulting in a significant number of dead cells in the final cell suspension. Subsequent analysis by flow cytometry is difficult to interpret due to non-specific binding of antibodies to dead cells and dead cell auto fluorescence. Factors released by dead cells can also interfere with downstream assays, complicating the study of primary tissues. During apoptosis, the cell membrane loses its phospholipid asymmetry resulting in exposure of negatively charged phospholipids on the cell surface. Relocation of phosphatidylserine (PS) to the outer leaflet of the cell membrane is a well-established marker of apoptosis. By targeting exposed PS with Annexin V we have developed a rapid method (EasySep™) to immunomagnetically remove dead cells from primary tissue samples. Performance of this kit was examined on various mouse and human tissue types. Using this method, we were able to improve viability of a single cell suspension of mouse lungs digested with collagenase/hyaluronidase from an initial viability of 39.6 ± 12.3% AnxV−/PI− to 70.9 ± 11.8% AnxV−/PI. From 1×10^8 total start cells, 1.43 ± 0.68 ×10^7 live cells were recovered (n=10). From human polymorphonuclear leukocytes cultured overnight, viability was improved from 23.7 ± 9.8% AnxV−/PI− to 67.7 ± 12.1% AnxV−/PI− with recovery of 1.27 ± 0.52 ×10^7 live cells from 1×10^8 total start cells (n=7). This equates to removal of 88.9 ± 8.3% and 91.8 ± 5.3% dead cells respectively. Since live cells are untouched, subsequent isolation of desired cell types can be performed, resulting in a more viable population of cells for downstream applications.

Group 2 innate lymphoid cells (ILC2s) have important roles in type-2 immune responses and are implicated in allergies, asthma, helminth infections and other metabolic diseases. To better understand their role in immunity, highly purified ILC2s are required. ILC2s are phenotypically defined as CD45+ lineage-negative CRTH2+ CD127+ CD161+, and comprise approximately 0.05% of peripheral blood mononuclear cells (PBMCs). Fluorescence activated cell sorting is the most common method to isolate ILC2s but it is both time-consuming and expensive because the cells are so rare. To address this issue, we have developed an immunomagnetic isolation method to obtain highly pure, particle-free ILC2s from human PBMCs in 2.5 hours. First, CRTH2 positive cells are labelled with a CRTH2-PE monoclonal antibody. These cells are then positively selected using an anti-PE antibody complex, EasySep™ Releasable RapidSpheres™ and an EasySep™ magnet. After separation, the magnetic particles are released from the positively selected cells and a second cocktail of antibody complexes and EasySep™ Dextran RapidSpheres™ is added to label non-ILC2s within the CRTH2 positive population. After a second separation, the released particles and unwanted cells are retained in the tube within the magnet, whereas the ILC2s are simply poured off and ready for use. With this method, the purity of ILC2s isolated from PBMCs was 84–95% (median 91%, n=18) with a recovery of 0.4–14.3×103 ILC2s per 108 starting PBMCs (median 2.46×103, n=18). The isolated ILC2s are functional, producing IL-13 upon in vitro stimulation with IL-33 and IL-2. Overall, EasySep™ Human ILC2 Isolation Kit allows researchers to easily and efficiently isolate highly pure and functional ILC2s.

MesenCult™-ACF-II is an improved animal component-free (ACF) culture medium for the derivation and expansion of mesenchymal progenitor cells (MPCs) from primary human bone marrow mononuclear cells (BMMC). This new medium is formulated to further enhance the expansion of MPCs obtained in a first generation medium, MesenCult™-ACF. To compare the performance of MesenCult™-ACF-II with MesenCult™-ACF, MPCs derived from primary BMMC were plated at 1–5 × 104 cells/cm2 in each formulation. Clonogenic growth was evaluated using the Colony-Forming Unit-Fibroblast (CFU-F) assay. To evaluate the expansion of MPC, BMMC were plated initially at 3–5 × 104 cells/cm2, and for subsequent subculture MPCs were plated at 1.5–3 × 103 cells/cm2 in each medium. The proliferative potential of MPCs was determined by counting the number of cells at each passage (P) up to P6, and paired t-test was used for statistical analysis. The generation of total CFU-F per 106 BMMC was comparable in the two media (71 ± 26 versus 69 ± 27 [mean ± SEM; n = 6] in MesenCult™-ACF- II and MesenCult™-ACF, respectively). The average fold-expansion of MPCs at each subculture was significantly higher in MesenCult™-ACF-II medium (10.7 ± 0.7 mean ± SEM; n = 9) than in MesenCult™-ACF (7.6 ± 0.3 mean ± SEM; n = 9; p < 0.05). Indeed, the expansion of MPCs in this new MesenCult™-ACF-II medium is as robust as that obtained in MesenCult™-XF (10.7 ± 0.7 versus 10.0 ± 0.6 [mean ± SEM; n = 9]; p > 0.05 in MesenCult™-ACF-II and MesenCult™-XF, respectively). The MPCs generated in MesenCult™-ACF-II can be cryopreserved in ACF Freezing Medium at any passage with high cell viability and recovery upon thawing (92.2% ± 2.9 and 74.8% ± 3.7 [mean ± SEM]; n = 3, viability and recovery, respectively). In summary, MesenCult™-ACF- II supports robust MPC expansion and together with the use of ACF Freezing Medium provides a complete ACF culture workflow for derivation, expansion and cryopreservation of these cells. Moreover, the MPCs cultured in MesenCult™-ACF-II differentiated robustly under the appropriate conditions into adipocytes, osteogenic cells and chondrocytes. Experiments to characterize MPCs cultured in MesenCult™-ACF-II medium from other (non-BM) tissue types are underway. The performance of MesenCult™-ACF-II medium is superior to that of the existing MesenCult™-ACF and equivalent to MesenCult™-XF.