Encapsulating Non-Human Primate Multipotent Stromal Cells in Alginate via High Voltage for Cell-Based Therapies and Cryopreservation

Alginate cell-based therapy requires further development focused on clinical application. To assess engraftment, risk of mutations and therapeutic benefit studies should be performed in an appropriate non-human primate model, such as the common marmoset (Callithrix jacchus). In this work we encapsulated amnion derived multipotent stromal cells (MSCs) from Callithrix jacchus in defined size alginate beads using a high voltage technique. Our results indicate that i) alginate-cell mixing procedure and cell concentration do not affect the diameter of alginate beads, ii) encapsulation of high cell numbers (up to 10×106 cells/ml) can be performed in alginate beads utilizing high voltage and iii) high voltage (15–30 kV) does not alter the viability, proliferation and differentiation capacity of MSCs post-encapsulation compared with alginate encapsulated cells produced by the traditional air-flow method. The consistent results were obtained over the period of 7 days of encapsulated MSCs culture and after cryopreservation utilizing a slow cooling procedure (1 K/min). The results of this work show that high voltage encapsulation can further be maximized to develop cell-based therapies with alginate beads in a non-human primate model towards human application.DFG/EXC/62/

Repeated freezing procedures preserve structural and functional properties of amniotic membrane for application in ophthalmology

For decades, the unique regenerative properties of the human amniotic membrane (hAM) have been successfully utilized in ophthalmology. As a directly applied biomaterial, the hAM should be available in a ready to use manner in clinical settings. However, an extended period of time is obligatory for performing quality and safety tests. Hence, the low temperature storage of the hAM is a virtually inevitable step in the chain from donor retrieval to patient application. At the same time, the impact of subzero temperatures carries an increased risk of irreversible alterations of the structure and composition of biological objects. In the present study, we performed a comprehensive analysis of the hAM as a medicinal product; this is intended for a novel strategy of application in ophthalmology requiring a GMP production protocol including double freezing– thawing cycles. We compared clinically relevant parameters, such as levels of growth factors and extracellular matrix proteins content, morphology, ultrastructure and mechanical properties, before and after one and two freezing cycles. It was found that epidermal growth factor (EGF), transforming growth factor beta 1 (TGF-ß1), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), hyaluronic acid, and laminin could be detected in all studied conditions without significant differences. Additionally, histological and ultrastructure analysis, as well as transparency and mechanical tests, demonstrated that properties of the hAM required to support therapeutic efficacy in ophthalmology are not impaired by dual freezing. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Human Amniotic Membrane: A review on tissue engineering, application, and storage

Human amniotic membrane (hAM) has been employed as scaffolding material in a wide range of tissue engineering applications, especially as a skin dressing and as a graft for corneal treatment, due to the structure of the extracellular matrix and excellent biological properties that enhance both wound healing and tissue regeneration. This review highlights recent work and current knowledge on the application of native hAM, and/or production of hAM-based tissue-engineered products to create scaffolds mimicking the structure of the native membrane to enhance the hAM performance. Moreover, an overview is presented on the available (cryo) preservation techniques for storage of native hAM and tissue-engineered products that are necessary to maintain biological functions such as angiogenesis, anti-inflammation, antifibrotic and antibacterial activity

Placenta and Placental Derivatives in Regenerative Therapies: Experimental Studies, History, and Prospects

Placental structures, capable to persist in a genetically foreign organism, are a natural model of allogeneic engraftment carrying a number of distinctive properties. In this review, the main features of the placenta and its derivatives such as structure, cellular composition, immunological and endocrine aspects, and the ability to invasion and deportation are discussed. These features are considered from a perspective that determines the placental material as a unique source for regenerative cell therapies and a lesson for immunological tolerance. A historical overview of clinical applications of placental extracts, cells, and tissue components is described. Empirically accumulated data are summarized and compared with modern research. Furthermore, we define scopes and outlooks of application of placental cells and tissues in the rapidly progressing field of regenerative medicine

Data from: Influence of factors of cryopreservation and hypothermic storage on survival and functional parameters of multipotent stromal cells of placental origin

Human placenta is a highly perspective source of multipotent stromal cells (MSCs) both for the purposes of patient specific auto-banking and allogeneic application in regenerative medicine. Implementation of new GMP standards into clinical practice enforces the search for relevant methods of cryopreservation and short-term hypothermic storage of placental MSCs. In this paper we analyze the effect of different temperature regimes and individual components of cryoprotective media on viability, metabolic and culture properties of placental MSCs. We demonstrate (I) the possibility of short-term hypothermic storage of these cells; (II) determine DMSO and propanediol as the most appropriate cryoprotective agents; (III) show the possibility of application of volume expanders (plasma substituting solutions based on dextran or polyvinylpyrrolidone); (IV) reveal the priority of ionic composition over the serum content in cryopreservation media; (V) determine a cooling rate of 1°C/min down to -40°C followed by immersion into liquid nitrogen as the optimal cryopreservation regime for this type of cells. This study demonstrates perspectives for creation of new defined cryopreservation methods towards GMP standards

Table 2. Cryopreservation of placental MSCs with various CPAs.

Data set for Table

Fig 1. Characterization of primary cell culture derived from placenta for MSC origin.

(A) RT-PCR analysis for expression of typical MSC markers: cells provide a signal for CD90, CD73, and CD105 MSC while being negative for CD34 hematopoietic marker. (B) Minus RT control for RT-PCR. (C,D) Cells display strong signals for CD73 and CD105 in flow cytometry analysis

Table 1. Characteristics of placental MSCs after short-term storage at different temperature conditions

Data set for Table

[Stade Pershing, 6/9/25,] Decaux [de Rouen, gagnant du 1000 m] : [photographie de presse] / [Agence Rol]

Référence bibliographique : Rol, 104062Appartient à l’ensemble documentaire : Pho20RolAppartient à l’ensemble documentaire : HNormand1Image de press

Fig 2. Differentiation potential of primary cell culture derived from placenta into lineages of MSC origin.

MSCs were differentiated into (A) adipogenic (Oil Red O staining), (B) osteogenic (Von Kossa staining), and (C) chondrogenic (Alcian blue staining) lineages as a proof of principle for plasticity of MSCs. Cells expanded in regular MSC culture medium represent the negative control (bar = 50 µm)