Evaluation of the biocompatibility of fish skin collagen with the mesenchymal stem cells in in vitro cultures.

There are scarce published data suggesting, that collagen extracted from fish skin may be an attractive alternative to mammalian-derived collagen for the in vitro cell cultures. In this study, we investigated proliferation potential and differentiation capability into osteogenic and adipogenic lineages of rat adipose-derived mesenchymal stem cells (rASCs) and human adipose-derived mesenchymal stem cells (hASCs) cultured on collagen extracted from silver carp and African sharptooth catfish skins, compared with commercially available mammalian collagen and collagen-free culture dishes. Our results revealed no significant differences between fish collagen and mammalian collagen in supporting cell viability and proliferation capacity. Fish-derived collagen is a cheap material derived from production waste, does not contain transmissible pathogens of mammalian origin, supports human cell cultures at comparable level to conventional collagen sources, and may be considered as the product of choice for the in vitro cell cultures.

Effect of adipose-derived stem cells seeding and surgical prefabrication on composite scaffold vascularization.

The study aimed to evaluate an angiogenic effect of adipose-derived stem cells (ASCs) seeding and surgical prefabrication (placing a vascular pedicle inside the scaffold) on developed composite scaffolds made of poly-ε-caprolactone (PCL), ß-tricalcium phosphate (ß-TCP), and poly (lactic-co-glycolic acid) (PLGA) (PCL+ß-TCP+PLGA). Moreover, we aimed to compare our data with previously tested PCL scaffolds to assess whether the new material has better angiogenic properties. The study included 18 inbred male WAG rats. There were three scaffold groups (six animals each): with non-seeded PCL+ß-TCP+PLGA scaffolds, with PCL+ß-TCP+PLGA scaffolds seeded with ASCs and with PCL+ß-TCP+PLGA scaffolds seeded with ASCs and osteogenic-induced. Each rat was implanted with two scaffolds in the inguinal region (one prefabricated and one non-prefabricated). After 2 months from implantation, the scaffolds were explanted, and vessel density was determined by histopathological examination. Prefabricated ASC-seeded PCL+ß-TCP+PLGA scaffolds promoted greater vessel formation than non-seeded scaffolds (19.73 ± 5.46 vs 12.54 ± 0.81; p = .006) and those seeded with osteogenic-induced ASCs (19.73 ± 5.46 vs 11.87±2.21; p = .004). The developed composite scaffold promotes vessel formation more effectively than the previously described PCL scaffold.

Chitosan-Based Dressing as a Sustained Delivery System for Bioactive Cytokines.

Wounds represent a common occurrence in human life. Consequently, scientific investigations are underway to advance wound healing methodologies, with a notable focus on dressings imbued with biologically active compounds capable of orchestrating the wound microenvironment through meticulously regulated release mechanisms. Among these bioactive agents are cytokines, which, when administered to the wound milieu without appropriate protection, undergo rapid loss of their functional attributes. Within the context of this research, we present a method for fabricating dressings enriched with G-CSF (granulocyte colony-stimulating factor) or GM-CSF (granulocyte-macrophage colony-stimulating factor), showcasing both biological activity and protracted release dynamics. Based on Ligasano, a commercial polyurethane foam dressing, and chitosan crosslinked with TPP (sodium tripolyphosphate), these dressings are noncytotoxic and enable cytokine incorporation. The recovery of cytokines from dressings varied based on the dressing preparation and storage techniques (without modification, drying, freeze-drying followed by storage at 4 °C or freeze-drying followed by storage at 24 °C) and cytokine type. Generally, drying reduced cytokine levels and their bioactivity, especially with G-CSF. The recovery of G-CSF from unmodified dressings was lower compared to GM-CSF (60% vs. 80%). In summary, our freeze-drying approach enables the storage of G-CSF or GM-CSF enriched dressings at 24 °C with minimal cytokine loss, preserving their biological activity and thus enhancing future clinical availability.

The impact of electroconductive multifunctional composite nanofibrous scaffold on adipose-derived mesenchymal stem cells.

BACKGROUND: The development of tissue-engineered scaffolds with electrical properties is the primary motivation of novel regenerative medicine. Electroconductive scaffolds are designed to mimic the injured tissue environment's electrical properties and regulate cellular behavior - growth, proliferation, and differentiation - that could stimulate the injured nerve's regeneration. METHODS: We fabricated dedicated electroconductive scaffolds and customized an appropriate device with an external current supply to expose cells on the scaffold to electrical stimulation (ES). Next, we isolated rat adipose-derived stem cells (ASCs) and performed in vitro experiments that combine cells, an electroconductive scaffold, NGF (nerve growth factor), and ES (90 mV/mm, constant, for four days). Finally, we checked cellular activity as proliferation, viability, morphology, the neurogenic differentiation potential of ASCs, cell alignment, and karyotype. RESULTS: We observed that the electrical stimulation did not change the viability and chromosome stability of rat ASCs, but altered slightly proliferation compared to non-stimulated cells. The combined effect of a scaffold, NGF, and ES caused morphology changes and enhancement of ASCs neuronal differentiation as indicated in ßIII-tubulin expression, actin organization, and upregulation of neurogenic gene expression. CONCLUSIONS: We developed an electroconductive scaffold and customized device for in vitro study with many experimental variants. Based on our results, we presumed that the established study scheme - including an electroconductive scaffold, NGF and ES - is biocompatible and could guide ASCs to differentiate in neurogenic lineage, thus may be potentially applied in nerve injury regeneration.

Encapsulation of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in liposomes prepared by thin film hydration and their transfer to mesenchymal stem cells and cord blood hematopoietic stem cells.

Introduction: Cytokines are important immune modulator factors controlling homeostasis of the body and are involved in tissue regeneration after wound healing. The encapsulation of cytokines in liposomes has many advantages potentially useful for their transfer to the cells. Liposomes protect cytokines from neutralization, improving their pharmacokinetics or biologic activity in vivo. They are targeted to specific cell types and may delay the release of cytokines, allowing their sustained paracrine delivery. Their physicochemical characteristics such as size, shape, charge, and stability are important parameters improving bio-distribution and prolonged pharmacokinetics of encapsulated cytokines. Material and methods: We developed an efficient protocol for the encapsulation of two types of cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), in liposomes that can be stored long term in the active state. Results: This method allows for the encapsulation of 12-13% of the total amount of cytokines and 50% of encapsulated cytokines are entrapped in liposomes of more than ≤ 600 nm in diameter. We show that in the studied cell lines the liposome-encapsulated cytokines do not affect cell morphology, proliferation or mortality. Conclusions: The G-CSF or GM-CSF can be delivered to the cells in working concentrations through the encapsulation in the liposomes. Before the clinical application, the efficiency of these liposomes should be confirmed by an in vivo study.

Perspectives for 3D-Bioprinting in Modeling of Tumor Immune Evasion.

In a living organism, cancer cells function in a specific microenvironment, where they exchange numerous physical and biochemical cues with other cells and the surrounding extracellular matrix (ECM). Immune evasion is a clinically relevant phenomenon, in which cancer cells are able to direct this interchange of signals against the immune effector cells and to generate an immunosuppressive environment favoring their own survival. A proper understanding of this phenomenon is substantial for generating more successful anticancer therapies. However, classical cell culture systems are unable to sufficiently recapture the dynamic nature and complexity of the tumor microenvironment (TME) to be of satisfactory use for comprehensive studies on mechanisms of tumor immune evasion. In turn, 3D-bioprinting is a rapidly evolving manufacture technique, in which it is possible to generate finely detailed structures comprised of multiple cell types and biomaterials serving as ECM-analogues. In this review, we focus on currently used 3D-bioprinting techniques, their applications in the TME research, and potential uses of 3D-bioprinting in modeling of tumor immune evasion and response to immunotherapies.

Modified Histopathological Protocol for Poly-ɛ-Caprolactone Scaffolds Preserving Their Trabecular, Honeycomb-like Structure.

Poly-ɛ-caprolactone (PCL) is now widely studied in relation to the engineering of bone, cartilage, tendons, and other tissues. Standard histological protocols can destroy the carefully created trabecular and honeycomb-like architecture of PCL scaffolds, and could lead to scaffold fibers swelling, resulting in the displacement or compression of tissues inside the scaffold. The aim of this study was to modify a standard histopathological protocol for PCL scaffold preparation and evaluate it on porous cylindrical PCL scaffolds in a rat model. In 16 inbred Wag rats, 2 PCL scaffolds were implanted subcutaneously to both inguinal areas. Two months after implantation, harvested scaffolds were first subjected to µCT imaging, and then to histopathological analysis with standard (left inguinal area) and modified histopathological protocols (right inguinal area). To standardize the results, soft tissue percentages (STPs) were calculated on scaffold cross-sections obtained from both histopathological protocols and compared with corresponding µCT cross-sections. The modified protocol enabled the assessment of almost 10× more soft tissues on the scaffold cross-section than the standard procedure. Moreover, STP was only 1.5% lower than in the corresponding µCT cross-sections assessed before the histopathological procedure. The presented modification of the histopathological protocol is cheap, reproducible, and allows for a comprehensive evaluation of PCL scaffolds while maintaining their trabecular, honeycomb-like structure on cross-sections.

In Vivo Supportive Effects of Mesenchymal Stem Cells on Fat Graft Stabilization and Local Induction of Angiogenesis Are Not Dependent on the Cell Donor Age or In Vitro Cell Culture Duration.

Mesenchymal stromal cells from adipose tissue (adipose stromal cells, ASCs) are regulators of repair processes in situ by paracrine mechanisms. These unique capabilities make ASCs candidates for the regenerative medicine applications, including cell-assisted lipotransfer method. ASC aging processes have been extensively researched in vitro, there is however limited information about the impact of ASC aging on their biological role in tissue regeneration in vivo. The aim of our study was the research of the possible effects of aging processes of ASCs resulting from the donor age or from in vitro aging during long-term culture (ASC expansion in bioreactors) on their capability to support survival of adipose subcutaneous transplants in rats. The supportive in vivo effects of ASCs from young donors were compared with the effects of ASCs from old donors and ASCs "aged" in long-term in vitro cultures. Fat grafts enriched with ASCs (regardless of their age) retain their volume longer than fat grafts without ASCs supplementation. Vascular expansion in cell-enriched fat grafts was more intense when compared with the controls. It may be concluded that the aging of ASCs does not substantially reduce their ability for the support of the survival of adipose tissue grafts.

Comparison of the Donor Age-Dependent and In Vitro Culture-Dependent Mesenchymal Stem Cell Aging in Rat Model.

Clinical experiments suggest that mesenchymal stem cells (MSCs) may be useful for tissue repair therapies or treatment of the autoimmune disorders. There is still lack of consensus concerning the age limit of MSC donors, majority of researchers suggest the autologous MSC therapies of patients not exceeding age limit of 55-60 yrs. The purpose of our study was to compare the selected parameters of MSCs from adipose tissue (adipose stem cell, ASC) collected from young and old rats of ages corresponding to patient's ages 25 yrs. and 80 yrs., respectively. The differences of parameters of ASCs from young and old animals were compared with the differences between ASCs from short-term (3 passage) and long-term (30 passage) in vitro culture. Cell morphology, surface marker expression, growth potential, metabolic activity, ß-galactosidase activity, clonogenic potential, angiogenic potential, and differentiation ability of ASCs from young and aged animals and from in vitro cultures at 3rd and 30th passages were compared and analyzed. It may be concluded that ASCs may be applied for autologous transplantations in aged patients. Comparison of ASC aging dynamics depending on host aging or in vitro culture duration suggests that long-term in vitro culture may affect ASCs more than natural aging process of their host. We suggest that ASCs expanded in vitro prior to their clinical use must be carefully screened for the possible aging effects resulting not only from donor age, but from the duration of their in vitro culture.

Bioactive Nanofiber-Based Conduits in a Peripheral Nerve Gap Management-An Animal Model Study.

The aim was to examine the efficiency of a scaffold made of poly (L-lactic acid)-co-poly(ϵ-caprolactone), collagen (COL), polyaniline (PANI), and enriched with adipose-derived stem cells (ASCs) as a nerve conduit in a rat model. P(LLA-CL)-COL-PANI scaffold was optimized and electrospun into a tubular-shaped structure. Adipose tissue from 10 Lewis rats was harvested for ASCs culture. A total of 28 inbred male Lewis rats underwent sciatic nerve transection and excision of a 10 mm nerve trunk fragment. In Group A, the nerve gap remained untouched; in Group B, an excised trunk was used as an autograft; in Group C, nerve stumps were secured with P(LLA-CL)-COL-PANI conduit; in Group D, P(LLA-CL)-COL-PANI conduit was enriched with ASCs. After 6 months of observation, rats were sacrificed. Gastrocnemius muscles and sciatic nerves were harvested for weight, histology analysis, and nerve fiber count analyses. Group A showed advanced atrophy of the muscle, and each intervention (B, C, D) prevented muscle mass decrease (p < 0.0001); however, ASCs addition decreased efficiency vs. autograft (p < 0.05). Nerve fiber count revealed a superior effect in the nerve fiber density observed in the groups with the use of conduit (D vs. B p < 0.0001, C vs. B p < 0.001). P(LLA-CL)-COL-PANI conduits with ASCs showed promising results in managing nerve gap by decreasing muscle atrophy.

Beneficial Effect of IL-4 and SDF-1 on Myogenic Potential of Mouse and Human Adipose Tissue-Derived Stromal Cells.

Under physiological conditions skeletal muscle regeneration depends on the satellite cells. After injury these cells become activated, proliferate, and differentiate into myofibers reconstructing damaged tissue. Under pathological conditions satellite cells are not sufficient to support regeneration. For this reason, other cells are sought to be used in cell therapies, and different factors are tested as a tool to improve the regenerative potential of such cells. Many studies are conducted using animal cells, omitting the necessity to learn about human cells and compare them to animal ones. Here, we analyze and compare the impact of IL-4 and SDF-1, factors chosen by us on the basis of their ability to support myogenic differentiation and cell migration, at mouse and human adipose tissue-derived stromal cells (ADSCs). Importantly, we documented that mouse and human ADSCs differ in certain reactions to IL-4 and SDF-1. In general, the selected factors impacted transcriptome of ADSCs and improved migration and fusion ability of cells in vitro. In vivo, after transplantation into injured muscles, mouse ADSCs more eagerly participated in new myofiber formation than the human ones. However, regardless of the origin, ADSCs alleviated immune response and supported muscle reconstruction, and cytokine treatment enhanced these effects. Thus, we documented that the presence of ADSCs improves skeletal muscle regeneration and this influence could be increased by cell pretreatment with IL-4 and SDF-1.

Scaffold vascularization method using an adipose-derived stem cell (ASC)-seeded scaffold prefabricated with a flow-through pedicle.

BACKGROUND: Vascularization is important for the clinical application of tissue engineered products. Both adipose-derived stem cells (ASCs) and surgical prefabrication can be used to induce angiogenesis in scaffolds. Our aim was to compare the angiogenic potential of ASC-seeded scaffolds combined with scaffold prefabrication with that of non-seeded, non-prefabricated scaffolds. METHODS: For prefabrication, functional blood vessels were introduced into the scaffold using a flow-through pedicle system. ASCs were isolated from rat fat deposits. Three-dimensional-printed cylindrical poly-ε-caprolactone scaffolds were fabricated by fused deposition modelling. Three groups, each containing six rats, were investigated by using non-seeded, ASC-seeded, and osteogenic induced ASC-seeded scaffolds. In each group, one rat was implanted with two scaffolds in the inguinal region. On the right side, a scaffold was implanted subcutaneously around the inferior epigastric vessels (classic prefabrication group). On the left side, the inferior epigastric vessels were placed inside the prefabricated scaffold in the flow-through pedicle system (flow-through prefabrication group). The vessel density and vascular architecture were examined histopathologically and by µCT imaging, respectively, at 2 months after implantation. RESULTS: The mean vessel densities were 10- and 5-fold higher in the ASC-seeded and osteogenic induced ASC-seeded scaffolds with flow-through prefabrication, respectively, than in the non-seeded classic prefabricated group (p < 0.001). µCT imaging revealed functional vessels within the scaffold. CONCLUSION: ASC-seeded scaffolds with prefabrication showed significantly improved scaffold vasculogenesis and could be useful for application to tissue engineering products in the clinical settings.

The Fluctuations of Leukocytes and Circulating Cytokines in Septic Humanized Mice Vary With Outcome.

Sepsis remains a major challenge in translational research given its heterogeneous pathophysiology and the lack of specific therapeutics. The use of humanized mouse chimeras with transplanted human hematopoietic cells may improve the clinical relevance of pre-clinical studies. However, knowledge of the human immuno-inflammatory response during sepsis in humanized mice is scarce; it is unclear how similar or divergent mouse and human-origin immuno-inflammatory responses in sepsis are. In this study, we evaluated the early outcome-dependent immuno-inflammatory response in humanized mice generated in the NSG strain after cecal ligation and puncture (CLP) sepsis. Mice were observed for 32 h post-CLP and were assigned to either predicted-to-die (P-DIE) or predicted-to-survive (P-SUR) groups for retrospective comparisons. Blood samples were collected at baseline, 6 and 24 h, whereas the bone marrow and spleen were collected between 24 and 32 h post-CLP. In comparison to P-SUR, P-DIE humanized mice had a 3-fold higher frequency of human splenic monocytes and their CD80 expression was reduced by 1.3-fold; there was no difference in the HLA-DR expression. Similarly, the expression of CD80 on the bone marrow monocytes from P-DIE mice was decreased by 32% (p < 0.05). Sepsis induced a generalized up-regulation of both human and murine plasma cytokines (TNFα, IL-6, IL-10, IL-8/KC, MCP-1); it was additionally aggravated in P-DIE vs. P-SUR. Human cytokines were strongly overridden by the murine ones (approx. ratio 1:9) but human TNFα was 7-fold higher than mouse TNFα. Interestingly, transplantation of human cells did not influence murine cytokine response in NSG mice, but humanized NSG mice were more susceptible to sepsis in comparison with NSG mice (79 vs. 33% mortality; p < 0.05). In conclusion, our results show that humanized mice reflect selected aspects of human immune responses in sepsis and therefore may be a feasible alternative in preclinical immunotherapy modeling.

Neurogenesis Using P19 Embryonal Carcinoma Cells.

The P19 cell line derived from a mouse embryo-derived teratocarcinoma has the ability to differentiate into the three germ layers. In the presence of retinoic acid (RA), the suspension cultured P19 cell line is induced to differentiate into neurons. This phenomenon is extensively investigated as a neurogenesis model in vitro. Therefore, the P19 cell line is very useful for molecular and cellular studies associated with neurogenesis. However, protocols for neuronal differentiation of P19 cell line described in the literature are very complex. The method developed in this study are simple and will play a part in elucidating the molecular mechanisms in neurodevelopmental abnormalities and neurodegenerative diseases.

Characterization and Optimization of the Seeding Process of Adipose Stem Cells on the Polycaprolactone Scaffolds.

The purpose of the current study was to evaluate the usefulness of adipose-derived stem cells (ASCs) for bone injury therapy. Lipoaspirates were collected from the abdomen regions of 17 healthy female donors (mean age 49 ± 6 years) using Coleman technique or Body-jet liposuction. In the present study, the primary objective was the in vitro characteristics of human ASCs. The secondary objective was the optimization of the cell seeding process on 3D-printed scaffolds using polycaprolactone (PCL) or polycaprolactone covered with tricalcium phosphate (PCL + 5% TCP). Biological evaluation of human ASC showed high efficiency of isolation obtaining a satisfying amount of homogeneous cell populations. Results suggest that ASCs can be cultured in vitro for a long time without impairing their proliferative capacity. Growth kinetics shows that the highest number of cells can be achieved in passage 5 and after the 16th passage; there is a significant decrease of cell numbers and their proliferative potential. The percentage of colony forming units from the adipose stem cells is 8% ± 0.63% (p < 0.05). It was observed that the accumulation of calcium phosphate in the cells in vitro, marked with Alizarin Red S, was increased along with the next passage. Analysis of key parameters critically related to the cell seeding process shows that volume of cell suspension and propagation time greatly improve the efficiency of seeding both in PCL and PCL + 5% TCP scaffolds. The cell seeding efficiency did differ significantly between scaffold materials and cell seeding methods (p < 0.001). Increased seeding efficiency was observed when using the saturation of cell suspension into scaffolds with additional incubation. Alkaline phosphatase level production in PCL + 5% TCP scaffold was better than in PCL-only scaffold. The study results can be used for the optimization of the seeding process and quantification methods determining the successful implementation of the preclinical model study in the future tissue engineering strategies.

Comparison of adipose stem cells sources from various locations of rat body for their application for seeding on polymer scaffolds.

Adipose tissue yields adult adipose stem cells (ASCs) in large quantities via less-invasive methods. These cells are of interest owing to their modulating properties and paracrine activities, which can be harnessed in regenerative medicine. Many studies on the use of rat fat tissue in an autologous animal model have been conducted; however, the different locations to obtain stromal vascular fraction of rat fat depots have not been fully characterized. The purpose of the current study was to identify optimal source of ASC from various locations of rat body. Animal experiments in vitro revealed that fat depots from cervical fat are an optimal ASC source. A high ASC yield facilitates subsequent studies on autologous transplantation in rats. The secondary objective was to compare the efficiency of osteoinductive media composition and evaluate of osteogenic potential of ASCs for seeding on scaffolds for bone repair. Scaffolds were assessed in vitro, using rat adipose stem cells and three-dimensional (3D) scaffolds comprising polycaprolactone (PCL) or polycaprolactone covered with tricalcium phosphate (PCL + 5%TCP). Seeded ASCs adhere to the surface and migrate to the scaffolds. Upon staining and determining alkaline phosphatase levels, PCL + 5%TCP scaffolds performed better than PCL scaffolds. Furthermore, growth factors such as BMP2 and FGF2 significantly increased ASC mineralization and induced osteogenesis (p < 0.05). Our results may help select and develop pre-clinical animal model for confirming the use of ASC, alone or in association with appropriate biomaterials for bone repair.

Safety of adipose-derived cell (stromal vascular fraction - SVF) augmentation for surgical breast reconstruction in cancer patients.

BACKGROUND: Progress in breast cancer surgery results in a decreased frequency of mastectomy, in the early phases of cancer replaced by breast conserving therapy (lumpectomy). Increased popularity of breast reconstruction by fat or adipose stem cells (ASC)-enriched fat transfer raised uncertainty about the possible risk of increased cancer recurrence. In vitro studies suggest that locally secreted cytokines and reconstructed local blood vessels may stimulate cancer expansion or cancer de novo induction from glandular tissue remaining after lumpectomy. OBJECTIVES: The purpose of the study was to evaluate the risk of cancer recurrence in breast cancer patients related to the stromal vascular fraction (SVF) augmentation during autologous fat grafting for breast reconstruction. MATERIAL AND METHODS: The tumor recurrence ratio in 56 patients having the breast reconstructed with autologous ASC (transplanted as the subpopulation present in SVF) was compared with the frequency of tumor recurrence in 252 matched patients treated in clinics without subsequent breast reconstruction. Adipose tissue was collected by the Coleman technique and split into 2 portions: one was used for breast reconstruction, the other was enzymatically digested, and isolated cells were used for the augmentation of fat implanted into the breast area. Cancer recurrence in the experimental and matched control group was evaluated following 3-year-long observation time, and the statistical significance of difference in cancer recurrence between the experimental and control group was evaluated. RESULTS: Cancer recurrence in the group of patients treated with ASC-enriched fat for breast reconstruction was 3.7% and did not differ significantly from the control group data (4.13%). No adverse effects of therapy were observed. CONCLUSIONS: Our study does not produce any data suggesting increased cancer risk following breast reconstruction after a mastectomy or a lumpectomy combined with local radiotherapy. It may be concluded that an autologous transplantation of fat augmented with ASC is a safe and efficient procedure. Longer observation time and the observation of larger numbers of patients would be useful for strengthening the conclusion.

Physical properties and biological interactions of liposomes developed as a drug carrier in the field of regenerative medicine.

Liposomes are used for encapsulation of the active compounds in different therapies, with the increasing frequency. The important areas of clinical applications of liposomes are cancer targeted treatment, antibiotic delivery or regenerative medicine. The liposomes can transfer both hydrophilic and hydrophobic compounds and have the lipid bilayer which imitates the cell membrane. Liposomes additionally may extend half-live period of drugs and protect them against the elimination in different ways, such as phagocytosis, enzymatic cleavage or exclusion by detoxification. The size and charge of liposomes play an important role in drug distribution and absorption into the cell. Limited data is available on the effects of liposomes on stem cells and progenitor cells. In this article, we examined the effect of charged conventional liposomes on growth of mesenchymal and blood stem cells isolated from umbilical cord. The data suggest a likelihood, that positively charged liposomes could impair stem cell growth and metabolism. Different methodological approaches allowed for the selection of negatively charged liposomes for further experiments, as the only type of liposomes which has the lowest cytotoxicity and does not affect hematopoietic cell proliferation.

Clinical Application of Autologous Adipose Stem Cells in Patients with Multiple Sclerosis: Preliminary Results.

The clinical outcome of autologous adipose stem cell (ASC) treatment of patients with multiple sclerosis (MS) was investigated following one year of observation. Methods. The clinical and MRI outcomes of 16 ASC-treated patients with RRMS and SPMS are reported after a one-year follow-up period. Results. At 18 months of follow-up, some patients showed "enticing" improvements on some exploratory efficacy measures, although a significant benefit was not observed for any measure across the entire group. Neither the progression of disability nor relapses were observed in any cases. In four patients, we found new gadolinium+ (Gd+) lesions on MRI. Our results indicate that ASC therapy is safe and does not produce any substantial side effects. Disease progression-free survival (PFS) of 18 months was seen in all patients with RRMS and SPMS. In these patients, EDSS scores did not progress above baseline scores. Gd-enhancing lesions were observed in two cases with RRMS, but these patients did not exhibit changes in EDSS score. Conclusion. Intrathecal treatment with ASCs is an attractive form of therapy for patients with MS but should be reserved for cases with aggressive disease progression, for cases that are still in the inflammatory phase, and for the malignant form.

Adipose-Derived Cells (Stromal Vascular Fraction) Transplanted for Orthopedical or Neurological Purposes: Are They Safe Enough?

Although mesenchymal stem cells are used in numerous clinical trials, the safety of their application is still a matter of concern. We have analysed the clinical results of the autologous adipose-derived stem cell treatment (stromal vascular fraction (SVF) containing adipose-derived stem cells, endothelial progenitors, and blood mononuclear cells) for orthopedic (cartilage, bone, tendon, or combined joint injuries) and neurologic (multiple sclerosis) diseases. Methods of adipose tissue collection, cell isolation and purification, and resulting cell numbers, viability, and morphology were considered, and patient's age, sex, disease type, and method of cell administration (cell numbers per single application, treatment numbers and frequency, and methods of cell implantation) were analysed and searched for the unwanted clinical effects. Results of cellular therapy were compared retrospectively to those obtained with conventional medication without SVF application. SVF transplantation was always the accessory treatment of patients receiving "standard routine" therapies of their diseases. Clinical experiments were approved by the Bioethical Medical Committees supervising the centers where patients were hospitalised. The conclusion of the study is that none of the treated patients developed any serious adverse event, and autologous mesenchymal stem (stromal) cell clinical application is a safe procedure resulting in some beneficial clinical effects (not analysed in this study).