An Approach towards a GMP Compliant In-Vitro Expansion of Human Adipose Stem Cells for Autologous Therapies

Human Adipose Tissue Stem Cells (hASCs) are a valuable source of cells for clinical applications (e.g., treatment of acute myocardial infarction and inflammatory diseases), especially in the field of regenerative medicine. However, for autologous (patient-specific) and allogeneic (off-the-shelf) hASC-based therapies, in-vitro expansion is necessary prior to the clinical application in order to achieve the required cell numbers. Safe, reproducible and economic in-vitro expansion of hASCs for autologous therapies is more problematic because the cell material changes for each treatment. Moreover, cell material is normally isolated from non-healthy or older patients, which further complicates successful in-vitro expansion. Hence, the goal of this study was to perform cell expansion studies with hASCs isolated from two different patients/donors (i.e., different ages and health statuses) under xeno- and serum-free conditions in static, planar (2D) and dynamically mixed (3D) cultivation systems. Our primary aim was I) to compare donor variability under in-vitro conditions and II) to develop and establish an unstructured, segregated growth model as a proof-of-concept study. Maximum cell densities of between 0.49 and 0.65 × 105 hASCs/cm2 were achieved for both donors in 2D and 3D cultivation systems. Cell growth under static and dynamically mixed conditions was comparable, which demonstrated that hydrodynamic stresses (P/V = 0.63 W/m3, τnt = 4.96 × 10−3 Pa) acting at Ns1u (49 rpm for 10 g/L) did not negatively affect cell growth, even under serum-free conditions. However, donor-dependent differences in the cell size were found, which resulted in significantly different maximum cell densities for each of the two donors. In both cases, stemness was well maintained under static 2D and dynamic 3D conditions, as long as the cells were not hyperconfluent. The optimal point for cell harvesting was identified as between cell densities of 0.41 and 0.56 × 105 hASCs/cm2 (end of exponential growth phase). The growth model delivered reliable predictions for cell growth, substrate consumption and metabolite production in both types of cultivation systems. Therefore, the model can be used as a basis for future investigations in order to develop a robust MC-based hASC production process for autologous therapies.

Effect of donor age and 3D-cultivation on osteogenic differentiation capacity of adipose-derived mesenchymal stem cells

Background and Purpose: Age and co-morbidities compromise healing tendencies of traumatic fractures in geriatric patients. Non-healing fractures may need regenerative medicine techniques involving autologous mesenchymal stem cells (MSCs). Donor age may affect the viability and differentiation capacity of MSCs. We investigated age-related differences in adipose-derived MSCs (AMSCs) concerning osteogenic potential in 2D and 3D cultivation. Materials and Methods: AMSCs were harvested from young (mean age: 37.5 ± 8.6 years) and old (mean age: 75.8 ± 9.2 years) patients. Cells were induced to osteogenic differentiation and cultivated in 2D and 3D for 14 days. Alkaline phosphatase (ALP) activity, mineralization and gene expression were investigated. Results: ALP activity revealed highest levels in 3D of old AMSCs after 14 days. ALP expression showed significant rises in old vs. young cells in 2D (p = 0.0024). Osteoprotegerin revealed the highest levels in old AMSCs in 2D. Highest osteocalcin levels presented in young cells compared to old cells in 2D (p = 0.0258) and young cells in 3D (p = 0.0014). Conclusion: 3D arrangement of old AMSCs without growth factors is not ensuring superior osteogenesis in vitro. AMSCs, especially cells from older patients, reveal higher osteogenic potential in 2D than in 3D. 3D arrangement favors osteogenic potential of young cells.

М3 макрофаги останавливают деление клеток предстательной железы больного раком простаты

В развитии рака предстательной железы (РПЖ) макрофаги играют важную роль. Многие опухоли выделяют противовоспалительные цитокины, которые перепрограммируют М1 фенотип макрофагов на проопухолевый М2 фенотип. М2 макрофаги подавляют противоопухолевый иммунитет, способствуют делению и метастазированию опухолевых клеток. Предыдущие исследования позволили нам обосновать предположение, что деление опухолевых клеток РПЖ человека может быть ограничено особым М3 фенотипом макрофагов. Фенотип М3, в отличие от М1 фенотипа, реагирует на противовоспалительные цитокины увеличением продукции провоспалительных противоопухолевых цитокинов, что способствует сохранению их противоопухолевых свойств в зоне опухоли. Цель исследования - проверка гипотезы о способности М3 макрофагов останавливать деление клеток предстательной железы больного РПЖ. Методика. В работе использовали макрофаги мышей, выделенные из перитонеального лаважа и макрофаги человека, полученные из моноцитов крови больных РПЖ. Фенотип М3 макрофагов получали добавлением в среду культивирования IFN-γ, ингибиторов STAT3, STAT6 и SMAD3 с последующей стимуляцией липополисахаридом. Результаты. Показано, что М3 макрофаги мышей и человека ограничивали деление клеток предстательной железы больных РПЖ в условиях 2D (на плоскости) культивирования на 43% и 93%, соответственно. При 3D (в объеме) культивировании М3 макрофаги мышей не ограничивали, а М3 макрофаги человека лишь незначительно ограничивали деление клеток предстательной железы у больных РПЖ. Заключение. Результаты работы делают обоснованными дальнейшие исследования и разработку клинической версии биотехнологии лечения рака предстательной железы с использованием М3 макрофагов.Macrophages play an important role in the development of prostate cancer (PCa). Many tumors, including PCa, secrete anti-inflammatory cytokines that reprogram the M1 macrophage phenotype into the pro-tumor M2 phenotype. M2 macrophages suppress antitumor immunity and promote division and metastasis of tumor cells. We hypothesized that the division of human PCa cells may be restricted by a specific M3 macrophage phenotype. The M3 phenotype, in contrast to the M1 phenotype, responds to anti-inflammatory cytokines by increasing the production of inflammatory anti-tumor cytokines and retains its anti-tumor properties in the tumor area. The aim of the study was to test the hypothesis on the ability of M3 macrophages to stop division of prostate cells from patients with PCa. Methods. This study used murine macrophages isolated from the peritoneal lavage and human macrophages obtained from blood monocytes of patients with PCa. The M3 macrophage phenotype was obtained by adding IFN-γ, STAT3, STAT6, and SMAD3 inhibitors to the cultural medium followed by lipopolysaccharide (LPS) stimulation. Results. Murine and human M3 macrophages restricted the division of patients’ PCa cells in the conditions of 2D cultivation by 43% and 93%, respectively. In 3D cultivation, murine M3 macrophages did not restrict whereas human M3 macrophages only slightly limited the division of prostate cells from PCa patients. The results of the study warrant further research and development of a clinical biotechnology for PCa treatment with reprogrammed M3 macrophages.

INTERACTION OF GOLD NANOPARTICLES WITH HUMAN HEPATOCYTES HEPG2 LINE UNDER CONDITIONS OF 2D- AND 3D-CULTIVATION

A previously undescribed phenomenon of the inability of cells to uptake gold nanoparticles (GNPs) was discovered when studying HepG2 cells under the conditions of 2D- and 3D- cultivation (monolayer and spheroids). The same cells readily internalized GNPs functionalized with polyethyleneimine.

Effect of Low-Molecular Fraction of Fish Peptide Albutrisan-NR on In Vitro Neurogenesis

The effect of the Albutrisan-NR substrate on the differentiation of the primary brain culture of aging rats and on the culture of the rabbit vibrissa dermal papilla was studied. The substance is a hydrolysate of fish tissue with a molecular weight of up to 5 kDa. It was found that adding 2% of the substance to the culture medium stimulates the growth and differentiation processes in rat brain cells. The possibility of using the substance as a growth factor for 3D cultivation of pluripotent dermal papilla cells and maintaining an uncommitted state was demonstrated. The use of Albutrisan-NR as a trophic factor in neuronal induction of dermal papilla cells was also shown.

3D culture of murine adipose-derived multipotent mesenchymal stromal cells in hydrogel based on carbomer 974P

Actual issues during tissue regeneration are to ensure the survival of transplanted cells at the site of their application and further activity, especially in case of local pathological alterations such as inflammation and ischemia. For this purpose, the matrices that can not only fill the defects of tissues, but also be scaffolds for cells are developed.The aim of this study was to evaluate the effectiveness of 3D cultivation of murine adipose-derived multipotent mesenchymal stromal cells (MSMCs) in hydrogel based on carbomer 974P.Materials and methods. MSMCs were obtained from the adipose tissue of FVB-Cg-Tg(GFPU)5Nagy/J mice transgenic for GFP gene. The cells were phenotyped by flow cytometry and directly differentiated into osteogenic and adipogenic direction to confirm multipotent phenotype. MMSCs were cultured and directly differentiated into osteogenic direction in three-dimensional hydrogel scaffolds. For hydrogel preparation we used carbomer 974P with composition of glycerol, propylene glycol, triethylamine and agarose in original proportion.Results. The three-dimensional hydrogel based on carbomer 974P for the further engraftment with MMSCs was obtained. Modified protocols for the preparation of hydrogels based on carbomer and agarose and their rehydration by culture media for the 3D cultivation of adipose-derived MMSCs have been developed. The optimal concentration of MSMCs and the injection method for engraftment of hydrogels of the required form and size are selected. It was shown that adipose-derived MMSCs in 3D carbomer hydrogel preserve the potential of directed osteogenic differentiation.Conclusion. Three-dimensional hydrogel based on carbomer 974P is capable to support cells, provide the necessary cytoarchitectonics, maintain intercellular interactions, which can promote further long-term survival and specialization of graft.

3D культура меланоцитов как тест-система и клеточная модель для изучения патологии меланогенеза

Функциональная активность меланоцитов обусловливает защитные свойства кожи против воздействия ультрафиолета, вызывающего фотостарение. Однако изучение меланоцитов in vivo затруднено тем, что необходимо использовать животные модели и трудоемкие методы анализа, а культивирование клеток в монослойной культуре in vitro сопряжено с потерей тканеспецифичных маркеров клеток. Данная работа посвящена получению и изучению сфероидов из меланоцитов, так как 3D культивирование меланоцитов в виде сфероидов может сохранить их фенотип и функциональность. Исследование проводили на первичной культуре меланоцитов кожи человека. Клетки культивировали в монослое в полной ростовой среде до 4 пассажа. Далее клетки помещали на агарозные планшеты с микролунками в посевной плотности 3,3 х 10 кл./мл. Анализ полученных сфероидов производили с помощью фотометрии, иммуноцитохимии и ПЦР в реальном времени. Было показано, что, при культивировании в монослое, к 4 пассажу снижалось количество синтезируемого меланина. Тогда как в 3D условиях меланоциты формировали компактные сфероиды, внутри которых в процессе культивирования не только сохранялся синтез, но и накапливался меланин. Была выявлена экспрессия специфических генов TYR и MCR1, и увеличивался синтез белков, участвующих в меланогенезе - gp100 и MITF. Таким образом, в данном исследовании было показано, что меланоциты in vitro способны формировать длительно живущие, жизнеспособные 3D структуры - сфероиды, с сохранением фенотипа и синтеза тканеспецифичных маркеров. Поэтому данные сфероиды могут быть успешно использованы как тест-системы для оценки эффективности препаратов, направленных на регуляцию уровня пигментации кожи. The functional activity of melanocytes determines protective properties of the skin against the effect of ultraviolet radiation, which causes photo-aging. However, studying melanocytes in vivo and in an in vitro monolayer culture is difficult because animal models and laborious analytical methods are required, and the culturing is associated with loss of tissue-specific cell markers. Since 3D cultivation of melanocytes in the form of spheroids can preserve their phenotype and functionality, this work focused on obtaining and studying melanocyte spheroids. The study was conducted using a primary culture of human skin melanocytes. Cells were cultured in a monolayer to the fourth passage. Then these cells were placed in agarose plates with microwells at the cell suspension concentration of 3.3 х 10 in vitro . These features make the melanocyte spheroids a convenient test-system for studying toxicity and efficiency of drugs targeted at regulation of skin pigmentation.