Questo lavoro è stato sostenuto dalla Fondazione per la ricerca austriaco (FWF, concedere N211-NAN per DS) e l&#39;Agenzia austriaca per la promozione della ricerca (FFG, concedere N200 a DS). Gli autori ringraziano URL Claudia per un&#39;eccellente assistenza tecnica e Monica Farrell per la modifica linguistica.

1. Materiale di partenza Inizia con plasma ricco di piastrine (PRP) unità preparata da cytapheresis o derivati ​​da buffy cappotti. 2. Sterilità controllo Per verificare la sterilità prendere un campione di 20 mL da ciascuna unità PRP, trasferendo il volume ad un sacchetto collegato piccolo (Baxter). Scollegare questa borsa mediante saldatura. 3. Congelamento delle unità di PRP Immediatamente dopo la preparazione, congelare le unità PRP fino ad almeno -20 ° C nel sacchetto originale senza ulteriori manipolazioni. 4. Scongelamento delle unità di HPL Quando i test batterici sono negativi, disgelo umano unità di lisato piastrinico, ora si chiama unità di HPL a 37 ° C (bagno d&#39;acqua) fino a quando i grumi di ghiaccio scompaiono. Non riscaldare il HPL. 5. Messa in comune delle unità di HPL Prendere almeno 10-15 unità scongelati HPL per un pool di lisato piastrinico per preparare un prodotto standardizzato. Collegare le sacche HPL consecutivamente per la borsa pooling doppio (MacoPharma) e trasferire la HPL in questi due sacchi. Scollegare le borse vuote HPL mediante saldatura. Ottenere un volume finale di 3 a 4 L di pool di lisato piastrinico umano (pHPL) mescolando il contenuto delle due borse. Collegare un piccolo sacchetto (Baxter) e prendere un campione di 20 mL pHPL per controllare la sterilità del prodotto in pool. Scollegare questa borsa mediante saldatura. 6. Porzionatura dei pHPL Parte il pHPL per ottenere aliquote adatto per ulteriori elaborazioni. Collegare sacchetti di piccole dimensioni (Baxter) per la borsa pooling doppio (Macopharma) e il trasferimento di volumi fino a 250 ml per le borse di storage di piccole dimensioni (Baxter). Scollegare i sacchi pieni di saldatura. 7. Re-congelamento delle aliquote pHPL Aumentare il tasso di frammentazione delle piastrine e la quantità di fattori di crescita rilasciati da un ulteriore gelo / disgelo passo. Congelare i sacchetti di pHPL porzionato di nuovo almeno -20 ° C. 8. Re-scongelamento e porzionatura del aliquote pHPL Scongelare le borse pHPL a 37 ° C (bagno d&#39;acqua). Trasferire il contenuto in flaconcini da 50 ml (Falcons BD) tagliando il tubo del sacchetto con le forbici sterili e versare il pHPL in fiale. Eseguire questo passaggio in un banco a flusso laminare per evitare contaminazioni batteriche o fungine. 9. Rimozione di frammenti di piastrine Come frammenti di piastrine tendono ad aggregarsi e possono indurre alloimmunizzazione, rimuoverli dalla pHPL. Centrifugare le provette pHPL quindi a 4.000 g (15 minuti, 4 ° C). In una pipetta da banco a flusso laminare il plasma surnatante in fiale stoccaggio finale (Falchi BD) e scartare il pellet di piastrine per evitare di frammenti in coltura cellulare. 10. Conservazione dei pHPL Congelamento aliquote di 50 ml fiale pHPL ancora almeno-20 ° C e conservarle per uso sperimentale. 11. L&#39;uso di pHPL in coltura cellulare Inizialmente aggiungere eparina senza conservanti al mezzo per evitare la formazione di gel. Scongelare un&#39;aliquota pHPL a 37 ° C e integrare il terreno di coltura con l&#39;aggiunta di 8 - 10%. Mezzi MSC-media: Utilizzare 500 ml di a-MEM, aggiungere 56 ml di scongelato pHPL (vedi anche riferimento 1 per ulteriori dettagli) e 2 IU / mL (= ml di soluzione madre 224) senza conservanti di eparina (evita la coagulazione del mezzo attraverso aggregazione di il fibrinogeno nel plasma) per raggiungere una concentrazione finale del 10% pHPL. Inoltre aggiungere penicillina (100U/mL) / streptomicina (100μg/mL) soluzione e 2mM di L-Glutamin (entrambi Sigma). Filtrare il medium attraverso un poro 20 micron filtro dimensioni del vuoto (Millipore). Etichetta della bottiglia appropriato (contenuto, data). ECFC-media: Utilizzare una bottiglia (500 ml) di EBM, aggiungere il citochine aliquote, 56 ml di pHPL, 10 UI / ml (= 1120μl di soluzione madre) di conservanti eparina, penicillina (100U/mL) / Streptomicina (100 g / mL) e 2mM soluzione di L-Glutamin al mezzo basale e il filtro con 20 l poro vuoto dimensione del filtro (Millipore). Etichetta della bottiglia appropriato (contenuto, data). <img src="/files/ftp_upload/1523/1523fig1.jpg" alt="Figura 1" /> Figura 1: Preparazione del plasma ricco di piastrine da una donazione di sangue intero da una banca del sangue locale o qualsiasi altro provider autorizzati. Dopo la centrifugazione del sangue può essere separato in plasma, buffy coat e globuli rossi. Quattro unità buffy coat, gruppo sanguigno O e un gruppo sanguigno AB plasma può essere raggruppati prima centrifugazione per separare il plasma ricco di piastrine. A regolare la qualità ricco di piastrine unità di plasma di circa 300mL dovrebbe contenere 1x10 9 piastrine per ml o 3x10 11 platelets totale. <img src="/files/ftp_upload/1523/1523fig2.jpg" alt="Figura 2" /><img src="/files/ftp_upload/1523/1523fig3.jpg" alt="Figura 3" />

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In alcune regioni plasma ricco di piastrine (PRP) possono essere ottenuti da buffy-coat altrimenti essendo un prodotto di scarto della confezionato produzione di globuli rossi da donazioni di sangue testato (Figura 1). In modo ottimale, PRP viene utilizzata immediatamente per l&#39;ulteriore preparazione di pHPL, come in piastrine superata concentra la disponibilità di fattori di crescita può essere ridotta a causa di lesioni di stoccaggio delle piastrine e la degradazione 20. Si raccomanda inoltre di produ...

<table><tbody><tr><td>Double bag (2 x 3.5L)</td><td>MacoPharma</td><td>VDL 8000XQ</td><td>originally for ascites puncture</td></tr><tr><td>50 mL Falcon tube</td><td>Falcon BD</td><td>2098</td><td></td></tr><tr><td>50 mL Stripettes</td><td>Costar</td><td>4501</td><td></td></tr><tr><td>Plasma bags (600mL)</td><td>Baxter Internationl Inc.</td><td>R4R2021</td><td></td></tr><tr><td>Sterile tubing welder</td><td>Terumo Medical Corp.</td><td>TSCD-II</td><td></td></tr><tr><td>Welding equipment </td><td>Fresenius Kabi</td><td>Compo Seal</td><td></td></tr><tr><td>Water bath</td><td></td><td></td><td></td></tr><tr><td>Sterile scissors</td><td></td><td></td><td></td></tr><tr><td>Clamps</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td></td></tr></tbody></table>

preparation of pooled human platelet lysate (phpl) as an efficient supplement for animal serum-free human stem cell cultures

Fattori di crescita derivato dalle piastrine hanno dimostrato di stimolare la proliferazione cellulare in modo efficiente<em&gt; In vivo</em<sup&gt; 1,2</sup&gt; E<em&gt; In vitro</em&gt;. Questo effetto è stato segnalato per le cellule stromali mesenchimali (MSCs), fibroblasti e endoteliali che formano colonie cellule con piastrine attivate da parte della trombina<sup&gt; 3-5</sup&gt; O lisate da cicli gelo / disgelo<sup&gt; 6-14</sup&gt; Prima del releasate piastrinica è aggiunto al mezzo di coltura cellulare. L&#39;effetto trofico dei fattori di crescita derivato dalle piastrine è già stato testato in studi diversi per l&#39;ingegneria dei tessuti e la terapia rigenerativa.<sup&gt; 1,15-17</supEfficienza&gt; Variando è considerato, almeno in parte a causa di concentrazioni singolarmente divergenti dei fattori di crescita<sup&gt; 18,19</sup&gt; E la mancanza attuale di protocolli standardizzati per la preparazione delle piastrine.<sup&gt; 15,16</sup&gt; Questo protocollo rappresenta una procedura praticabile per generare un pool di risorse umane lisato piastrinico (pHPL) derivati ​​da plasma regolarmente prodotto ricco di piastrine (PRP) del 40-50 donazioni di sangue singolo. Da diversi cicli gelo / disgelo le membrane delle piastrine sono danneggiati e fattori di crescita sono efficacemente rilasciati nel plasma. Infine, i frammenti delle piastrine vengono rimossi mediante centrifugazione per evitare la formazione di ampi aggregati e impoveriscono gli antigeni potenziale. L&#39;attuazione di pHPL in protocolli standard di cultura rappresenta uno strumento promettente per l&#39;ulteriore sviluppo di terapie cellulari propagato in un animale privo di proteine ​​del sistema.

Watch this Scientific Journal Video about Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures at JoVE.com

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Umano lisato piastrinico è una ricca fonte di fattori di crescita e di un supplemento potente nelle cellule in coltura. Questo protocollo presenta il processo di preparazione di un ampio pool di lisato piastrinico umano partendo da plasma ricco di piastrine, eseguendo numerosi cicli di gelo-disgelo e riducono i frammenti delle piastrine.

Preparazione del pool lisato piastrinico umani (pHPL) come supplemento efficiente per animali siero-Free Culture sulle cellule staminali umane

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

preparation-pooled-human-platelet-lysate-phpl-as-an-efficient

Research

JoVE Journal

Biology

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

25.1K Views.  Medical University of Graz, Austria. Umano lisato piastrinico è una ricca fonte di fattori di crescita e di un supplemento potente nelle cellule in coltura. Questo protocollo presenta il processo di preparazione di un ampio pool di lisato piastrinico umano partendo da plasma ricco di piastrine, eseguendo numerosi cicli di gelo-disgelo e riducono i frammenti delle piastrine.

Video: Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, underlies the only known cure of human immunodeficiency virus (HIV-1) infection, and shows immense promise in the treatment of genetic diseases such as beta thalassemia. Our group has developed a protocol to model HSPC gene therapy in nonhuman primates (NHPs), allowing scientists to optimize many of the same reagents and techniques that are applied in the clinic. Here, we describe methods for purifying CD34+ HSPCs and long-term persisting hematopoietic stem cell (HSC) subsets from primed bone marrow (BM). Identical techniques can be employed for the purification of other HSPC sources (e.g., mobilized peripheral blood stem cells [PBSCs]). Outlined is a 2 day protocol in which cells are purified, cultured, modified with lentivirus (LV), and prepared for infusion back into the autologous host. Key readouts of success include the purity of the CD34+ HSPC population, the ability of purified HSPCs to form morphologically distinct colonies in semisolid media, and, most importantly, gene modification efficiency. The key advantage to HSPC gene therapy is the ability to provide a source of long-lived cells that give rise to all hematopoietic cell types. As such, these methods have been used to model therapies for cancer, genetic diseases, and infectious diseases. In each case, therapeutic efficacy is established by enhancing the function of distinct HSPC progeny, including red blood cells, T cells, B cells, and/or myeloid subsets. The methods to isolate, modify, and prepare HSPC products are directly applicable and translatable to multiple diseases in human patients.

The goal of this protocol is to isolate nonhuman primate CD34+ cells from primed bone marrow, to gene-modify these cells with lentiviral vectors, and to prepare a product for infusion into the autologous host. The total protocol length is approximately 48 h.

Preparazione e modificazione genetica di staminali ematopoietiche Nonhuman Primate e cellule progenitrici

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, ...

Ricerca

Genetica

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.&#160; Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

Produzione senza siero di epofere umane tridimensionali da cellule staminali pluripotenti

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human ...

Biologia dello sviluppo

Preparing Activated Platelet-Rich Plasma for Culturing Human Adipose-Derived Stem Cells

Activated platelet-rich plasma (PRP) prepared from whole blood via centrifugation demonstrated a proliferation-stimulating effect in several kinds of cultured cells, implying a possible use in regenerative medicine. Here, a double-spin method was used to prepare PRP from whole blood. PRP was further activated by autologous thrombin. The platelet count was measured in the activated PRP and the proliferation-stimulating effect in human adipose-derived stem cells (hASCs) was examined. The resulting platelet count was 11.5-times higher in PRP than in whole blood plasma. The proliferation of hASCs was markedly enhanced by incubation with 1% PRP. The described method can be used to reproducibly prepare PRP with a high concentration of platelets. PRP prepared by this method markedly promotes proliferation of hASCs.

In this report, we describe a double-spin method for preparing activated platelet-rich plasma (PRP). Using autologous thrombin, human adipose-derived stem cells (hASCs) were cultured. Activated PRP was shown to promote proliferation of hASCs.

Activated platelet-rich plasma (PRP) prepared from whole blood via centrifugation demonstrated a proliferation-stimulating effect in several kinds of ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Derivazione libero-Feeder della cresta neurale progenitrici Cellule pluripotenti da cellule staminali umane

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Neuroscienze

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

Cultura di cellule staminali pluripotenti umane il polivinil alcool-Co-itaconico acido idrogel con diverse rigidità nelle circostanze senza Xeno

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

Bioingegneria

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

Analisi di flusso laminare-based per indagare il reclutamento leucocitario su cellule vascolari coltivate e piastrine aderenti

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

Immunologia e infezioni

Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not represent the underlying cytoarchitectural and functional complexity of the affected individuals carrying suspected disease variants. Conventional 2-D models remain incomplete representations of in vivo-like structures and do not adequately capture the complexity of the brain. Thus, there is an emerging need for more 3-D hiPSC-based models that can better recapitulate the cellular interactions and functions seen in an in vivo system.
Here we report a protocol to develop a 3-D system from undifferentiated hiPSCs based on the serum free embryoid body (SFEB). This 3-D model mirrors aspects of a developing ventralized neocortex and allows for studies into functions integral to living neural cells and intact tissue such as migration, connectivity, communication, and maturation. Specifically, we demonstrate that the SFEBs using our protocol can be interrogated using physiologically relevant and high-content cell based assays such as calcium imaging, and multi-electrode array (MEA) recordings without cryosectioning. In the case of MEA recordings, we demonstrate that SFEBs increase both spike activity and network-level bursting activity during long-term culturing. This SFEB protocol provides a robust and scalable system for the study of developing network formation in a 3-D model that captures aspects of early cortical development.

Here we report a protocol to develop a three-dimensional (3-D) system from human induced-pluripotent stem cells (hiPSCs) called the serum free embryoid body (SFEB). This 3-D model can be used like an organotypic slice culture to model human cortical development and for the physiological interrogation of developing neural circuits.

Sviluppare corpi embrioni liberi del siero derivato da HiPSC per l&#39;interrogazione di colture a cellule staminali a 3 dosi utilizzando saggi fisiologicamente rilevanti

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

Generazione di organoidi dei vasi sanguigni umani da cellule staminali pluripotenti

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

Isolamento e coltura di cellule staminali umane derivate da adiposio con un innovativo metodo xenogenico-free per la terapia umana

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...

Medicina

Un sistema epatico 2D+ completamente umano fisiologicamente rilevante per la scoperta di farmaci

An All-Human Hepatic Culture System for Drug Development Applications

Finding a long-term, human-relevant culture model for primary human hepatocytes (PHHs) for pharmacological and toxicological studies remains a challenge. Current in vitro model platforms are often inconvenient and complex, lack phenotypic stability over time, and do not support multiple PHH lots, lacking experimental reproducibility and flexibility. Here, we provide a detailed protocol for the thawing, plating, and maintenance of an all-human 2D+ hepatic system (TV2D+), which takes advantage of standard two-dimensional (2D) culture techniques and equipment while maintaining the longevity and phenotypic stability over time that typically accompany more complex three-dimensional (3D) systems. The results show attachment and percent plateability in TV2D+ as a function of PHH seeding density, as well as stable functionality for at least 2 weeks in culture. A range of PHH seeding densities are assessed to achieve a successful long-term culture. When established properly, the PHHs in TV2D+ organize into hepatocyte colonies, express a hepatic-specific marker, and maintain viability, architectural integrity, and physiologically relevant levels of albumin and urea. This unique combination of attributes makes the TV2D+ system a suitable hepatic model for a variety of pharmacological and toxicological applications.

Autore in primo piano: Sviluppo di un modello epatico semplice e robusto per applicazioni farmacologiche e tossicologiche 

Recent technical advances enabled the scaled production of an in vitro platform for drug metabolism and toxicity applications. An all-human 2D+ hepatic system (TV2D+) provides physiologically relevant results using traditional two-dimensional culture methods. This protocol will support end-users in the setup, maintenance, and application of the system.

Un sistema di coltura epatica interamente umano per applicazioni di sviluppo di farmaci

Finding a long-term, human-relevant culture model for primary human hepatocytes (PHHs) for pharmacological and toxicological studies remains a challenge. ...