Autologous Human Serum Research Articles

Characteristics of Buccal Fat Adipose-Derived Stem Cells Cultured in Autologous Human Serum- and Fetal Bovine Serum-Supplemented Media: A Comparative Study

Characteristics of Buccal Fat Adipose-Derived Stem Cells Cultured in Autologous Human Serum- and Fetal Bovine Serum-Supplemented Media: A Comparative Study

Peptide ligands on the PEGylated nanoparticle surface and human serum composition are key factors for the interaction between immune cells and nanoparticles

The architecture of a nanoparticles’ surface formed due to a modification with a ligand and protein corona formation in biofluids is critical for interactions with cells in vivo. Here we studied interactions of immune cells with magnetic nanoparticles (MNPs) covalently modified with polyethylene glycol (PEG) and their counterparts conjugated with peptides: a pH (low) insertion peptide (pHLIP) and cycloRGD as a targeting ligand in human serum. The conjugation of MNPs-PEG with pHLIP, but not with cycloRGD, enhanced the association of these particles with mononuclear phagocytic cells in vitro and in vivo. We did not find a clear difference in protein corona composition between the pHLIP-modified and parental PEGylated nanoparticles. Analysis of the effect of autologous human serum on MNP uptake by monocytes showed that the efficiency of endocytosis varies among healthy donors and depends on intrinsic properties of serum. Nevertheless, using classic blood, coagulation, biochemical tests, and anti-PEG IgG serum level, we failed to identify the cause of the observed interdonor variation. These individual differences should be taken into consideration during testing of nanotherapeutics.

Human Platelet Lysate as an Alternative to Autologous Serum for Human Chondrocyte Clinical Use.

ObjectiveA pivotal aspect of cartilage tissue engineering resides in cell culture medium supplementation, in view of maximizing in vitro cell proliferation and preserving cellular functionality. Autologous human serum (aHS) is commonly used as an inducive supplement for safe human articular chondrocyte (HAC) proliferation prior to clinical implantation. However, practical clinical use of aHS is hindered by constraining manufacturing requirements and quality assurance-driven downstream processing. The present study investigated potential alternative use of commercial human platelet lysate (hPL) supplements in HAC manufacturing workflows related to clinical therapeutic pathways.DesignDifferential effects of hPL, aHS, and fetal bovine serum were assessed on primary cultured HAC parameters (viability, proliferative rates, and morphology) in 2-dimensional in vitro systems. A 3-dimensional HAC pellet model served for postexpansion assessment of cellular functionality, by visualizing proteoglycan production (Alcian blue staining), and by using qRT-PCR relative quantification of chondrogenic marker (SOX9, COL2-A1, and ACAN) genetic expression.ResultsWe found that monolayer HAC culture with hPL or aHS supplements presented similar characteristics (elongated cell morphology and nearly identical growth kinetics). Chondrogenic activity appeared as conserved in HACs expanded with human or bovine supplements, wherein histologic analysis indicated a progressive sGAG accumulation and SOX9, COL2-A1, ACAN gene expression was upregulated in 3-dimensional HAC pellet models.ConclusionThis study therefore supports the use of hPL as a functional equivalent and alternative to aHS for cultured HAC batch preparation, with the potential to effectively alleviate pressure on clinical and manufacturing bottlenecks in cell therapy approaches for cartilage regeneration.

Clinically compatible advances in blood-derived endothelial progenitor cell isolation and reprogramming for translational applications

The promise of using induced pluripotent stem cells (iPSCs) for cellular therapies has been hampered by the lack of easily isolatable and well characterized source cells whose genomes have undergone minimal changes during their processing. Blood-derived late-outgrowth endothelial progenitor cells (EPCs) are used for disease modeling and have potential therapeutic uses including cell transplantation and the translation of induced pluripotent stem cell (iPSC) derivatives. However, the current isolation of EPCs has been inconsistent and requires at least 40−80 mL of blood, limiting their wider use. In addition, previous EPC reprogramming methods precluded the translation of EPC-derived iPSCs to the clinic. Here a series of clinically-compatible advances in the isolation and reprogramming of EPCs is presented, including a reduction of blood sampling volumes to 10 mL and use of highly efficient RNA-based reprogramming methods together with autologous human serum, resulting in clinically relevant iPSCs carrying minimal copy number variations (CNVs) compared to their parent line.

Immunotherapy response modeling by ex-vivo organ culture for lung cancer.

One of the major hurdles for the advancement of cancer immunotherapy is lack of robust, accessible experimental models. We aimed to produce an ex-vivo organ culture (EVOC) model of immunotherapy for non-small cell lung cancer (NSCLC). Freshly resected early stage tumors were collected from the operating room, fragmented to clusters < 450µm and cultured with fetal calf serum and human autologous serum. The resulting EVOC includes cancer epithelial cells within tumor tissue clusters and immune cells. Original tissue features are reflected in the EVOCs. The response to immune checkpoint inhibitors (ICI) was assessed by IFNγ gene induction. Interestingly, IFNγ EVOC induction was numerically higher when anti-CTLA4 was added to anti-PD-L1 treatment, supporting the notion that anti-CTLA4 impacts cancer partly through tumor-resident immune cells. In parallel, immunohistochemistry (IHC) for key immune-related proteins was performed on the formalin-fixed paraffin embedded (FFPE) corresponding tumors. EVOC IFNγ induction by ICI correlated with basal non-induced IFNγ, CD8, CD4 and FOXP3 mRNA levels within EVOCs and with tumor-FFPE-IHC for CD8 and granzyme B. A weaker correlation was seen with tumor-FFPE-IHC for CD3, CD4, CD68, FOXP3 and tumor-PD-L1. Tertiary lymphoid structure density was also correlated with the ICI response. Our study provides novel data about biomarkers that correlate with ICI-induced response of early stage NSCLC. Retention of the microenvironment and minimal addition of exogenous factors suggest this model to reliably represent the original tumor. The cluster-based EVOC model we describe can provide a valuable, yet simple and widely applicable tool for the study of immunotherapy in NSCLC.

Comparative characteristics of monocytes isolation conditions by adhesion method for in vitro experiments

Introduction . Monocytes and macrophages are cells that play an important role in the function of the innate immune system and may be involved in the pathological process in various diseases. For this reason, in vitro studies of the functional activity of these cells are of great scientific interest. The literature indicates a large number of methods used to isolate primary monocytes but the approaches are often controversial. Aim . To perform a comparative analysis of the influence of various conditions on monocytes adhesion efficiency to determine the most optimal combinations of factors providing the highest yield and purity of the obtained cells. Materials and methods . Peripheral venous blood samples were obtained from 6 healthy volunteers. Peripheral blood mononuclear cells (PBMC) were isolated by standard centrifugation on Ficoll density gradient (1.077 g/ml). After the centrifugation, PBMC were plated in wells of a 24-well plate, coated with collagen or uncoated, and incubated for 2 or 24 hours in RPMI-1640 medium containing 10% fetal calf (FCS) or autologous human serum (AHS). The number of attached monocytes was assessed by epifluorescence microscopy after staining with Hoechst 33342 with automatic counting of nuclei in ten fields of view. Results . It has been established that incubation of cells for 2 hours in wells without coating in the presence of 10% FCS allows obtaining the largest number of monocytes with median purity of 80.9 (66.5-89.1)%. The number of monocytes obtained by adhesion in collagen-coated wells tended to be lower, regardless of the type of serum used. In our experiment, the adhesion capacity of monocytes was reduced when cells were plated in uncoated wells with 10% AHS. Extended to 24 hours incubation time was accompanied by a significant decrease in the number of attached monocytes in most cases. Conclusion . Incubation of PBMC for 2 hours on uncoated tissue culture plastic in the presence of 10% FCS provides the highest yield and purity of monocytes in comparison with other tested conditions.

Abstract 700: Recellularization of Acellular Xenogenic Scaffold With Autologous Human Mesenchymal Stem Cells Rescues the Xenoreactive Immune Response

Background: Pre-clinical evidence suggests that structural valve deterioration (SVD) of bioprosthetic xenogenic tissue heart valves (XTHV) is a result of chronic immune rejection. Recellularization of XTHVs with human autologous cells represents one potential strategy to prevent this xenoreactive immune response and mitigate SVD. Methods: Bone marrow, human pericardium and heparinized whole blood was collected from adult patients undergoing elective cardiac surgery. Decellularized bovine pericardium underwent recellularization with hMSCs, which were isolated and cultured from the bone marrow, via co-incubation to allow for cell seeding. To examine the cell mediated immune response, we performed immunofluorescent staining for CD68+ macrophages and CD3+ T-cells and performed a proliferation assay to quantify T-cell proliferation. To investigate the humoral immune response, we performed an enzyme linked immunosorbent assay for pro-inflammatory cytokines TNF-α and INF-γ. Serum and PBMCs were collected for subsequent biochemical and flow cytometric analysis. Results: We show that decellularized bovine pericardium, exposed to human serum, had significantly decreased T-cell activation, represented by TNF-αand INF-γexpression, as well as a significant decrease in T-cell proliferation, when compared to wild-type bovine pericardium (p&lt;0.01). Moreover, when decellularized bovine pericardium was recellularized with autologous hMSCs and exposed to human serum, there was an additional decrease in TNF-α, INF-γexpression along with a further significant decrease in T-cell proliferation, when compared to both wild-type and decellularized bovine pericardium (p&lt;0.01). Importantly, recellularized XTHVs exposed to human serum had an equivalent expression of TNF-α, INF-γ and T-cell proliferation when compared to native human pericardium exposed to autologous human serum. Conclusions: Taken together, our data suggest that autologous human MSC recellularization of decellularized bovine pericardium abrogates the xenoreactive immune response. As such, autologous hMSC recellularization of an acellular xenogenic scaffolds may be an effective approach to decrease the progression of bioprosthetic SVD.

Differential influences of Fc gamma receptor blocking on the effects of certolizumab pegol and infliximab on human monocytes

Objectives: To compare the effects of certolizumab pegol (CZP) and infliximab (IFX) on human monocytes.Methods: Highly purified monocytes from healthy donors were cultured with CZP, IFX, control IgG1, or polyethylene glycol (PEG) at pharmacological attainable concentrations in culture medium with 10% autologous normal human serum (NHS) or with fetal bovine serum (FBS) for 24 h, after which the supernatants were replaced by fresh culture medium containing LPS. After additional 24 h of incubation, the supernatants were assayed for TNF-α and IL-6. In some experiments, the cells were harvested after 1 h of stimulation with LPS for analysis of mRNA for TNF-α by quantitative PCR.Results: Pre-incubation of monocytes with CZP or IFX reduced the production of TNF-α in subsequent cultures stimulated by LPS in a dose-dependent manner. The suppressive effects of IFX on the TNF-α production were significantly diminished, but those of CZP were rather enhanced, in cultures with autologous NHS compared with in cultures with FBS. Addition of IgG, but not IgG F(ab′)2 fragments, significantly inhibited the suppressive effects of IFX on the production of TNF-α and IL-6, whereas either IgG or IgG F(ab′)2 fragments had no significant influences on the suppressive effects of CZP. Furthermore, pre-incubation with CZP or IFX significantly inhibited the expression of mRNA for TNF-α and IL-6 in monocytes compared with PEG or IgG.Conclusion: These results indicate that the mechanism of action of CZP is different from that of IFX. Thus, CZP suppresses the production of proinflammatory cytokines independently of Fc receptors, whereas the suppressive effects of IFX on human monocytes are almost totally dependent on the interaction with Fc receptors.

Membrane complement regulatory protein reduces the damage of transplanting autologous bone marrow mesenchymal stem cells by suppressing the activation of complement

There are few studies on the interaction of transplanting autologous bone marrow mesenchymal stem cells (BMSCs) and complement. In order to further explore the effect of complement on BMSCs, BMSCs were obtained from bone marrow of 20 cases clinical patients, and then experimented in vitro. The cytotoxicity of complement on the mesenchymal stem cells in autologous human serum (AHS) was measured by Europium cytotoxicity assay. The complement membrane attack complex (MAC) deposited on the membrane surface was detected by flow cytometry. Finally, the cytotoxicity on BMSCs was measured after mCRPs overexpression or knockdown. We found that more than 90% of cells derived from bone marrow were identified to be mesenchymal stem cells through detection of cell membrane surface markers by flow cytometry. BMSCs harvested from the 20 patients all had cytotoxicity after incubated with AHS, and the cytotoxicity was significant higher than that incubated with complement inactivated autologous human serum (iAHS). Complement attack complex (MAC) could be detected on the BMSCs incubated with AHS, which implied the complement activation. We also found that mCRPs CD55 and CD59 overexpressions can resist the cytotoxicity induced by complement activation, while mCRPs CD55 and CD59 knockdown can enhance the cytotoxicity. Thus, the results indicated that mCRPs could effectively protect BMSCs from attacking by complement by suppressing the activation of complement.

Autologous serum supplement favours in vitro regenerative paracrine factors synthesis.

Foetal bovine serum (FBS) is often the serum supplement of choice for in vitro human cell culture. This study compares the effect of FBS and autologous human serum (AuHS) supplement in human peripheral blood mononuclear cell (PBMC) culture to prepare secretome. The PBMC (n=7) were cultured either in RPMI-1640 containing L-glutamine and 50units/ml Penicillin-Streptomycin (BM) or in BM with either AuHS or FBS. Viability, proliferation and differentiation of PBMC were evaluated. Paracrine factors present in the secretomes (n=6) were analysed using ProcartaPlex Human Cytokine panel (17 plex). Ingenuity Pathway Analysis (IPA) was performed to predict activation or inhibition of biological functions related to tissue regeneration. The viability of PBMC that were cultured with FBS supplement was significantly reduced at 96h compared to those at 0 and 24h (P<.05). While the reduction of the viability of PBMC that were cultured with AuHS supplement was not significantly different compared to those at 0 and 24h. The FBS secretomes prepared at 24h was found to contain significantly higher amount of EGF (P<.05) compared to that in AuHS or BM secretome. The AuHS secretomes contained significantly higher amount of HGF at 24 (P<.05) and 96h (P<.01), and VEGF-A at 24h (P<.05) compared to those in the FBS secretomes. SDF-1 was not detected in the FBS secretomes prepared at either 24 or 96hours. Double immunocytochemical staining revealed a marked increase in co-localization of SDF-1 and its receptor in PBMC that were cultured with AuHS supplement compared to that cultured with FBS supplement. In secretome preparation, AuHS supplement favours synthesis of paracrine factors that are needed for regenerative therapy.

Effect of human autologous serum and fetal bovine serum on human corneal epithelial cell viability, migration and proliferation in vitro.

To analyze the concentration-dependent effects of autologous serum (AS) and fetal bovine serum (FBS) on human corneal epithelial cell (HCEC) viability, migration and proliferation. AS was prepared from 13 patients with non-healing epithelial defects Dulbecco's modified eagle medium/Ham's F12 (DMEM/F12) with 5% FBS, 0.5% dimethyl sulphoxide (DMSO), 10 ng/mL human epidermal growth factor, 1% insulin-transferrin-selenium, then were incubated in serum media: DMEM/F12 supplemented by 5%, 10%, 15% or 30% AS or FBS. HCEC viability was analyzed using cell proliferation kit XTT, migration using a wound healing assay, proliferation by the cell proliferation enzyme-linked immunosorbent assay (ELISA) BrdU kit. Statistical analysis was performed using the generalized linear model, the values at 30% AS or 30% FBS were used as the baselines. HCEC viability was the highest at 30% AS or 15% FBS and the lowest at 10% AS or 30% FBS application. HCEC migration was the quickest through 30% AS or 30% FBS and the slowest through 5% AS or 5% FBS concentrations. Proliferation was the most increased through 15% AS or 5% FBS and the least increased through 30% AS or 30% FBS concentrations. HCEC viability at 10% and 15% AS was significantly worse (P=0.001, P=0.023) compared to baseline and significantly better at 15% FBS (P=0.003) concentrations. HCEC migration was significantly worse (P≤0.007) and HCEC proliferation significantly better (P<0.001) in all concentration groups compared to baseline. For the best viability of HCEC 30% AS or 15% FBS, for HCEC migration 30% AS or 30% FBS, for proliferation 15% AS or 5% FBS should be used. Therefore, we suggest the use of 30% AS in clinical practice.

Oral mucosa: an alternative epidermic cell source to develop autologous dermal-epidermal substitutes from diabetic subjects.

Oral mucosa has been highlighted as a suitable source of epidermal cells due to its intrinsic characteristics such as its higher proliferation rate and its obtainability. Diabetic ulcers have a worldwide prevalence that is variable (1%-11%), meanwhile treatment of this has been proven ineffective. Tissue-engineered skin plays an important role in wound care focusing on strategies such autologous dermal-epidermal substitutes. Objective The aim of this study was to obtain autologous dermal-epidermal skin substitutes from oral mucosa from diabetic subjects as a first step towards a possible clinical application for cases of diabetic foot.Material and Methods Oral mucosa was obtained from diabetic and healthy subjects (n=20 per group). Epidermal cells were isolated and cultured using autologous fibrin to develop dermal-epidermal in vitro substitutes by the air-liquid technique with autologous human serum as a supplement media. Substitutes were immunocharacterized with collagen IV and cytokeratin 5-14 as specific markers. A Student´s t- test was performed to assess the differences between both groups.Results It was possible to isolate epidermal cells from the oral mucosa of diabetic and healthy subjects and develop autologous dermal-epidermal skin substitutes using autologous serum as a supplement. Differences in the expression of specific markers were observed and the cytokeratin 5-14 expression was lower in the diabetic substitutes, and the collagen IV expression was higher in the diabetic substitutes when compared with the healthy group, showing a significant difference.Conclusion Cells from oral mucosa could be an alternative and less invasive source for skin substitutes and wound healing. A difference in collagen production of diabetic cells suggests diabetic substitutes could improve diabetic wound healing. More research is needed to determine the crosstalk between components of these skin substitutes and damaged tissues.

Abstract 615: Scaffold-based perfusion bioreactor system for in vitro maintenance of primary breast cancer tissue microenvironment suitable for personalized medicine

Abstract INTRODUCTION Two-dimensional (2D) in vitro culture systems and in vivo animal models are the primary tools used to test cancer cell responses to drugs but they are not suited for the development of immune-mediate therapies. Here we present an innovative method for in vitro culture primary breast cancer (BrCa) tissues in porous 3D scaffolds by using a perfusion-based bioreactor system (U-CUP). MATERIALS &amp; METHODS Freshly excised breast cancer specimens were fragmented and cultured in a 3D “sandwich-like format” between collagen porous scaffolds under perfusion flow. DMEM/F12, supplemented with 10% autologous human serum, was used as a culture medium. Malignant and non-malignant cells survival, expansion into the scaffold and the ability to recapitulate features of the original BrCa specimens were histologically assessed. For estrogen receptor (ER) positive tissues we tested the response to hormonal therapy by adding the anti-ER drug Fulvestrant. Furthermore, the maintenance of immune-infiltrating cells allowed testing immune blockade therapy in vitro using anti PD-L1 on PD-L1 positive samples. Response to treatment was evaluated by histology and qRT-PCR for markers of immune-response. RESULTS By culturing BrCa using the U-CUP we were able to preserve viability and to promote the expansion of breast cancer cells from surgical specimens together with accompanying stromal and immune cells into the porous scaffold. Expanding cancer cells were viable after 21 days and recapitulating the initial histology with formation of glands. Administration of anti-ER treatment was associated with decreased expansion of cancer tissue into the scaffold after 21 days. After 7 days of anti PD-L1 antibody treatment we observed a reduced number of tumor cells due to the activation of infiltrating lymphocytes, as shown by increased expression of IFNg and decreased expression of IL10. CONCLUSIONS The scaffold-based perfusion bioreactor represents a successful organotypic tumor model allowing in vitro long-term culture of breast cancer specimens. Our findings shed the light on a promising system for selecting personalized treatment based on a patient's tumor specific microenvironment. Citation Format: Manuele Giuseppe Muraro, Simone Muenst, Valentina Mele, Luca Quagliata, Alexandar Tzankov, Walter P. Weber, Giulio C. Spagnoli, Savas D. Soysal. Scaffold-based perfusion bioreactor system for in vitro maintenance of primary breast cancer tissue microenvironment suitable for personalized medicine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 615.

The role of membrane complement regulatory protein in transplantation of bone marrow mesenchymal stem cell

Objective To explore the effect of complement activation on bone marrow mesenchymal stem cells (BMSCs) and evaluate the effect after transfection of complement regulatory proteins. Methods Bone marrow aspirate was harvested from 10 cases of patients suffered from fractures. Mesenchymal stem cells were isolated, indentified cultured and then experimented in vitro. The complement cytotoxicity on the mesenchymal stem cells in autologous serum was measured by Europium cytotoxicity assay. The samples were divided into BMSCs group, BMSCs+ autologous human serum (AHS) group and BMSCs+ inactivated autologous human serum (iAHS) group. The complement membrane attack complex (MAC) deposited on the membranes was detected by flow cytometry. Finally, the cytotoxicity on BMSCs was measured after transfected with membrane complement regulatory proteins (mCRPs). All samples were divided into BMSCs with mCRPs untransfected group and BMSCs with mCRPs transfected group. Results More than 95% of cells derived from bone marrow were identified to be mesenchymal stem cells through detection of cell surface markers by flow cytometry. The cytotoxicity of untreated cells was 0.41%±1.48%. BMSCs harvested from the 10 patients all had cytotoxicity after incubated with autologous serum, and the cytotoxicity was 32.59%±2.73%, while cytotoxicity after incubated with complement inactivated autologous serum was 2.59%±3.08%, which was similar to control group. Complement attack complex (MAC) could be detected on the BMSCs incubated with autologous serum, which implied the complement activation. After transfection of mCRPs, the cytotoxicity of autologous serum on transfected cells was decreased. The cytotoxicity of untransfected cells (41.70%±4.47%) had significant difference compared to the cells transfected with CD55 (21.87%±2.19%), the cells transfected with CD59 (18.67%±1.42%), and the cells transfected with CD46+CD55+CD59 (28.43%±2.14%). CD55, CD59 and CD46+ CD55+CD59 transfected groups could impair effectively the cytotoxicity from complement. However, the cytotoxicity impairment was less effective in CD46 transfected cells (39.30%±3.96%), which had no significant difference compared to untransfected cells. Conclusion Membrane complement regulatory proteins could effectively protect bone marrow mesenchymal stem cells from attacks by complement. Key words: Bone marrow; Mesenchymal stem cells; Complement membrane attack complex

Abstract P4-04-19: Primary breast cancer culture in a perfusion-based bioreactor suitable for in vitro testing of immune blockade therapy

Abstract Introduction: Interaction between cancer cells and immune system critically affects development, progression and treatment of human malignancies. Two-dimensional (2D) in vitro culture systems and in vivo animal models are the primary tools used to test cancer cell response to drugs but they are not suited for the development of immune-mediated therapies. Here we present an innovative method to culture breast cancer tissue in porous 3D scaffolds by using a perfusion-based bioreactor system that allows the maintenance and expansion of tumor microenvironment. Experimental procedures: Freshly excised breast cancer specimens were fragmented and cultured in a 3D "sandwich-like format" between two layers of porous collagen scaffold under perfusion flow (U-CUP). DMEM/F12, supplemented with 10% autologous human serum, was used as a culture medium. We assessed the ability of tumor and non-malignant cells to survive and expand into the scaffold in perfusion culture, as well as their capacity to recapitulate features of the original breast cancer tissue. The maintenance of immune-infiltrating cells allowed testing of immune blockade therapy in vitro using anti-PD-L1 and anti-CTLA4 antibodies alone or in combination. Results: The U-CUP culture system preserved tissue viability better compared to a static culture and promoted the expansion of breast cancer cells from surgical specimens together with accompanying stromal and immune cells into the porous scaffold. Tumor tissues were viable after 21 days and mostly recapitulating the initial histology with formation of glands. Administration of anti-PDL1 antibody, alone or in combination with anti-CTLA4, to the culture medium was associated with increased expression of markers of immune-activation (i.e. IFNg) and decreased expression of immunosuppressive cytokine IL10. Conclusions: Our results show that culture of breast cancer tissue in a 3D perfusion-based bioreactor might represent a promising system for the pre-clinical evaluation of immune-mediated therapies. Preserving malignant, interstitial and immunocompetent cells comprised in surgically excised breast cancer samples might allow a direct evaluation of the effects of various treatments on the complex tumor microenvironment. This engineered in vitro model could be extended as a platform allowing the testing of innovative approaches for the treatment of human malignancies, possibly in the direction of personalized medicine. Citation Format: Muraro MG, Muenst S, Mele V, Spagnoli GC, Oertli D, Weber WP, Soysal SD. Primary breast cancer culture in a perfusion-based bioreactor suitable for in vitro testing of immune blockade therapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-19.

Platelet-derived growth factor (PDGF)-AA/AB in human serum are potential indicators of the proliferative capacity of human synovial mesenchymal stem cells.

IntroductionFor expansion of human mesenchymal stem cells (MSCs), autologous human serum is safer than fetal bovine serum in clinical situations. One of the problems with the use of autologous human serum is that its proliferative effect on MSCs varies widely between donors. The threefold goals of this study were: (1) to demonstrate an improved method for preparing human serum; (2) to identify growth factors predictive of proliferative potential; and (3) to identify a cytokine to promote MSC proliferation in human serum.MethodsFresh blood was collected using a closed bag system containing glass beads. The bag was shaken at 20 °C for 30 minutes for rapid preparation, or kept stationary at 4 °C for 24 hours for slow preparation. Passage 0 synovial MSCs derived from four donors were cultured with 10 % conventional rapid preparation serum or modified slow preparation serum from four different donors. To perform the colony-forming unit assay, synovial MSCs were cultured in these serums. The protein expression profile in serum was analyzed using cytokine array. The candidate proteins were speculated from the correlation between the colony-forming ability and protein expression. As an evaluation of the candidate proteins, proliferation ability, surface marker phenotype and differentiation capability of synovial MSCs were examined.ResultsCompared with rapid preparation serum, slow preparation serum resulted in a significantly higher total colony number and twofold higher expression levels of nine proteins (angiopoietin-1, BDNF, EGF, ENA-78, IGFBP-2, platelet-derived growth factor (PDGF)-AA, PDGF-AB/BB, RANTES and TfR). Colony number was positively correlated with PDGF-AA/AB concentrations. Exogenous PDGF-AA significantly promoted proliferation of synovial MSCs, whereas PDGF receptor (PDGFR) inhibitor decreased it. Addition of PDGFs or PDGFR inhibitor did not affect surface epitopes of synovial MSCs. Pretreatment with PDGFs or PDGFR inhibitor did not affect chondrogenic, adipogenic, or calcification potentials of synovial MSCs.ConclusionSlow preparation serum contained higher concentrations of PDGF-AA/AB and increased the colony formation number of synovial MSCs. PDGF-AA/AB were indicators of the proliferative potential of human serum. Exogenous PDGF-AA increased proliferation of synovial MSCs without alteration of surface epitopes and differentiation potentials.

Tooth Tissue Engineering: The Importance of Blood Products as a Supplement in Tissue Culture Medium for Human Pulp Dental Stem Cells.

One of the goals in using cells for tissue engineering (TE) and cell therapy consists of optimizing the medium for cell culture. The present study compares three different blood product supplements for improved cell proliferation and protection against DNA damage in cultured human dental pulp stem cells for tooth TE applications. Human cells from dental pulp were first characterized as adult stem cells (ectomesenchymal mixed origin) by flow cytometry. Next, four different cell culture conditions were tested: I, supplement-free; II, supplemented with fetal bovine serum; III, allogeneic human serum; and IV, autologous human serum. Cultured cells were then characterized for cell proliferation, mineralized nodule formation, and colony-forming units (CFU) capability. After 28 days in culture, the comet assay was performed to assess possible damage in cellular DNA. Our results revealed that Protocol IV achieved higher cell proliferation than Protocol I (p = 0.0112). Protocols II and III resulted in higher cell proliferation than Protocol I, but no statistical differences were found relative to Protocol IV. The comet assay revealed less cell damage in cells cultured using Protocol IV as compared to Protocols II and III. The damage percentage observed on Protocol II was significantly higher than all other protocols. CFUs capability was highest using Protocol IV (p = 0.0018) and III, respectively, and the highest degree of mineralization was observed using Protocol IV as compared to Protocols II and III. Protocol IV resulted in significantly improved cell proliferation, and no cell damage was observed. These results demonstrate that human blood product supplements can be used as feasible supplements for culturing adult human dental stem cells.

Abstract 305: An innovative 3D porous scaffold-based perfusion bioreactor system for the in vitro maintenance and expansion of primary breast cancer tissue

Abstract Introduction: Development of cancer treatment protocols has traditionally been based on studies in experimental animal models or taking advantage of human established tumor cell lines for in vitro and in vivo assays. Here we present an innovative method to culture breast cancer tissues in porous 3D scaffolds by using a perfusion-based bioreactor system. Experimental procedures: Freshly excised estrogen-receptor positive breast cancer specimens were fragmented and cultured in a 3D “sandwich-like format” between porous scaffolds under perfusion flow, with “on-line” monitoring of environmental condition. DMEM/F12, supplemented with 10% autologous human serum, was used as a culture medium in the presence or absence of estrogens. The ability of tumor cells to survive and expand into the scaffold in perfusion culture as well as their ability to recapitulate features of the original breast cancer specimens was histologically assessed. Results: With this innovative method we were able to preserve viability and to promote the expansion of breast cancer cells from surgical specimens together with accompanying stromal and immune cells into the porous scaffold. Expanding cancer cells were viable after 21 days and recapitulating the initial histology with formation of glands. Administration of estrogen to the culture medium was associated with increased expansion of cancer cells into the scaffold starting at 14 days of culture. Conclusions: Culture of breast cancer tissue in a 3D scaffold-based bioreactor preserves malignant, interstitial and immunocompetent cells present in surgically excised breast cancer samples, thus allowing direct testing of basic cancer research hypotheses in intact, complex human tumor microenvironments. Furthermore, it might allow a direct evaluation of the effects of various treatments on malignant and non-transformed cells. This engineered in vitro model could be extended beyond the context of our primary scientific interest, namely breast cancer, as a broader platform allowing animal-free testing of innovative approaches for the treatment of human malignancies, possibly in the direction of personalized medicine. Citation Format: Manuele G. Muraro, Simone Muenst, Giulio C. Spagnoli, Daniel Oertli, Walter P. Weber, Savas D. Soysal. An innovative 3D porous scaffold-based perfusion bioreactor system for the in vitro maintenance and expansion of primary breast cancer tissue. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 305. doi:10.1158/1538-7445.AM2015-305

Serum and supplement optimization for EU GMP‐compliance in cardiospheres cell culture

Cardiac progenitor cells (CPCs) isolated as cardiospheres (CSs) and CS-derived cells (CDCs) are a promising tool for cardiac cell therapy in heart failure patients, having CDCs already been used in a phase I/II clinical trial. Culture standardization according to Good Manufacturing Practices (GMPs) is a mandatory step for clinical translation. One of the main issues raised is the use of xenogenic additives (e.g. FBS, foetal bovine serum) in cell culture media, which carries the risk of contamination with infectious viral/prion agents, and the possible induction of immunizing effects in the final recipient. In this study, B27 supplement and sera requirements to comply with European GMPs were investigated in CSs and CDCs cultures, in terms of process yield/efficiency and final cell product gene expression levels, as well as phenotype. B27− free CS cultures produced a significantly reduced yield and a 10-fold drop in c-kit expression levels versus B27+ media. Moreover, autologous human serum (aHS) and two different commercially available GMP AB HSs were compared with standard research-grade FBS. CPCs from all HSs explants had reduced growth rate, assumed a senescent-like morphology with time in culture, and/or displayed a significant shift towards the endothelial phenotype. Among three different GMP gamma-irradiated FBSs (giFBSs) tested, two provided unsatisfactory cell yields, while one performed optimally, in terms of CPCs yield/phenotype. In conclusion, the use of HSs for the isolation and expansion of CSs/CDCs has to be excluded because of altered proliferation and/or commitment, while media supplemented with B27 and the selected giFBS allows successful EU GMP-complying CPCs culture.

Expansion of Semi-Automatic Processed Human Adipose-Derived Stem Cells in Medium Supplemented with Autologous Serum and Antioxidants

Objective: Excised fat tissue (EFT) is an excellent source of adipose-derived stem cells (ADSCs) for autologous cell therapy. However, the current technique for excising fat tissues with conventional surgical scissors and using culture medium containing fetal bovine serum (FBS) may not comply with Good Tissue Practice (GTP) guidelines. We thus developed a semi-automated process complying with the abovementioned guideline to manufacture clinicalgrade ADSCs from small volumes of EFT. Methods: A semi-automated process was developed by using a disposable disperser tube operated on a Tube Disperser Workstation to obtain stromal vascular fraction (SVF) from EFT. A culture medium supplemented with human autologous serum (HAS) and mesenchymal stem cell culture adjuvant (MCA) consisted of basic fibroblast growth factor (bFGF/FGF-2) and antioxidants, was formulated to expand ADSCs from SVF in vitro. Results: The semi-automated process can markedly enhance cell yields and reduce the operation time for mincing tissues from an hour down to a few minutes. The growth of ADSCs was slower in the 10% HAS medium than that in medium supplemented with FBS, but their growth in the 10% HAS medium containing MCA (10% HAS+MCA) was superior to both media. The 10% HAS+MCA medium also promoted adipogenesis, osteogenesis, chondrogenesis and increased the expression of CD44, CD73 and the stemness genes in the ADSCs. Conclusion: Our novel semi-automated process provides an efficient isolation of SVF, and the 10% HAS+MCA medium accelerates the growth of ADSCs from small amounts of EFT without losing their differentiation ability.