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Diffuse high-grade gliomas contain some of the most dangerous human cancers that lack curative treatment options. The recent molecular stratification of gliomas by the World Health Organisation in 2021 is expected to improve outcomes for patients in neuro-oncology through the development of treatments targeted to specific tumour types. Despite this promise, research is hindered by the lack of preclinical modelling platforms capable of recapitulating the heterogeneity and cellular phenotypes of tumours residing in their native human brain microenvironment. The microenvironment provides cues to subsets of glioma cells that influence proliferation, survival, and gene expression, thus altering susceptibility to therapeutic intervention. As such, conventional in vitro cellular models poorly reflect the varied responses to chemotherapy and radiotherapy seen in these diverse cellular states that differ in transcriptional profile and differentiation status. In an effort to improve the relevance of traditional modelling platforms, recent attention has focused on human pluripotent stem cell-based and tissue engineering techniques, such as three-dimensional (3D) bioprinting and microfluidic devices. The proper application of these exciting new technologies with consideration of tumour heterogeneity and microenvironmental interactions holds potential to develop more applicable models and clinically relevant therapies. In doing so, we will have a better chance of translating preclinical research findings to patient populations, thereby addressing the current derisory oncology clinical trial success rate.
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Neoplasias Encefálicas , Glioma , Humanos , Neoplasias Encefálicas/patologia , Glioma/patologia , Diferenciação Celular , Microambiente TumoralRESUMO
This report summarizes the recent activity of the International Stem Cell Banking Initiative held at Harvard Stem Cell Institute, Boston, MA, USA, on June 18, 2017. In this meeting, we aimed to find consensus on ongoing issues of quality control (QC), safety, and efficacy of human pluripotent stem cell banks and their derivative cell therapy products for the global harmonization. In particular, assays for the QC testing such as pluripotency assays test and general QC testing criteria were intensively discussed. Moreover, the recent activities of global stem cell banking centers and the regulatory bodies were briefly summarized to provide an overview on global developments and issues. Stem Cells 2019;37:1130-1135.
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Células-Tronco Pluripotentes/citologia , Células-Tronco/citologia , Bancos de Tecidos/normas , Boston , Terapia Baseada em Transplante de Células e Tecidos/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Cooperação Internacional , Controle de QualidadeRESUMO
Electrical stimulation of hydrogels has been performed to enable micro-actuation or controlled movement of ions and biomolecules such as in drug release applications. Hydrogels are also increasingly used as low modulus, biocompatible coatings on electrode devices and thus are exposed to the effects of electrical stimulation. As such, there is growing interest in the latter, especially on the dynamic and nanoscale physical properties of hydrogels. Here, we report on the electro-mechano properties of photocrosslinkable gelatin methacrylate (GelMA) hydrogel applied as coatings on conducting polymer polypyrrole-dodecylbenze sulfonate (PPy-DBSA) electrodes. In particular, Electrochemical-Atomic Force Microscopy (EC-AFM) was used to quantify the nanoscale actuation and dynamic changes in Young's modulus as the GelMA coating was electrically stimulated via the underlying PPy-DBSA electrode. Pulsed electrical stimulation was shown to induce dynamic expansion and contraction, or nanoscale actuation, of the GelMA hydrogel due to the reversible ingress of electrolyte ions and associated changes in osmotic pressure during oxidation and reduction of the PPy-DBSA film. In addition, dynamic changes in the Young's modulus of up to 50% were observed in the hydrogel and correlated with the actuation process and ion diffusion during oxidation and reduction of the underlying PPy-DBSA film. These dynamic properties were investigated for hydrogels with varying degrees of cross-linking, porosity and modulus, the latter ranging from ≈0.2-1 kPa. The study demonstrates an AFM-based approach to quantify the dynamic physical properties of hydrogels, which are shown to be modulated via electrical stimulation. This can enable a better understanding of the electro-mechano mechanisms that are important for the controlled release of drugs or controlling cell interactions at the hydrogel-cell interface.
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The present work describes a preclinical trial (in silico, in vivo and in vitro) protocol to assess the biomechanical performance and osteogenic capability of 3D-printed polymeric scaffolds implants used to repair partial defects in a sheep mandible. The protocol spans multiple steps of the medical device development pipeline, including initial concept design of the scaffold implant, digital twin in silico finite element modeling, manufacturing of the device prototype, in vivo device implantation, and in vitro laboratory mechanical testing. First, a patient-specific one-body scaffold implant used for reconstructing a critical-sized defect along the lower border of the sheep mandible ramus was designed using on computed-tomographic (CT) imagery and computer-aided design software. Next, the biomechanical performance of the implant was predicted numerically by simulating physiological load conditions in a digital twin in silico finite element model of the sheep mandible. This allowed for possible redesigning of the implant prior to commencing in vivo experimentation. Then, two types of polymeric biomaterials were used to manufacture the mandibular scaffold implants: poly ether ether ketone (PEEK) and poly ether ketone (PEK) printed with fused deposition modeling (FDM) and selective laser sintering (SLS), respectively. Then, after being implanted for 13 weeks in vivo, the implant and surrounding bone tissue was harvested and microCT scanned to visualize and quantify neo-tissue formation in the porous space of the scaffold. Finally, the implant and local bone tissue was assessed by in vitro laboratory mechanical testing to quantify the osteointegration. The protocol consists of six component procedures: (i) scaffold design and finite element analysis to predict its biomechanical response, (ii) scaffold fabrication with FDM and SLS 3D printing, (iii) surface treatment of the scaffold with plasma immersion ion implantation (PIII) techniques, (iv) ovine mandibular implantation, (v) postoperative sheep recovery, euthanasia, and harvesting of the scaffold and surrounding host bone, microCT scanning, and (vi) in vitro laboratory mechanical tests of the harvested scaffolds. The results of microCT imagery and 3-point mechanical bend testing demonstrate that PIII-SLS-PEK is a promising biomaterial for the manufacturing of scaffold implants to enhance the bone-scaffold contact and bone ingrowth in porous scaffold implants. MicroCT images of the harvested implant and surrounding bone tissue showed encouraging new bone growth at the scaffold-bone interface and inside the porous network of the lattice structure of the SLS-PEK scaffolds.
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Materiais Biocompatíveis , Mandíbula , Alicerces Teciduais , Animais , Ovinos , Mandíbula/cirurgia , Mandíbula/diagnóstico por imagem , Alicerces Teciduais/química , Impressão Tridimensional , Análise de Elementos Finitos , OsteogêneseRESUMO
The periosteum is a thin layer of connective tissue covering bone. It is an essential component for bone development and fracture healing. There has been considerable research exploring the application of the periosteum in bone regeneration since the 19th century. An increasing number of studies are focusing on periosteal progenitor cells found within the periosteum and the use of hydrogels as scaffold materials for periosteum engineering and guided bone development. Here, we provide an overview of the research investigating the use of the periosteum for bone repair, with consideration given to the anatomy and function of the periosteum, the importance of the cambium layer, the culture of periosteal progenitor cells, periosteum-induced ossification, periosteal perfusion, periosteum engineering, scaffold vascularization, and hydrogel-based synthetic periostea.
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Autologous bone replacement remains the preferred treatment for segmental defects of the mandible; however, it cannot replicate complex facial geometry and causes donor site morbidity. Bone tissue engineering has the potential to overcome these limitations. Various commercially available calcium phosphate-based bone substitutes (Novabone®, BioOss®, and Zengro®) are commonly used in dentistry for small bone defects around teeth and implants. However, their role in ectopic bone formation, which can later be applied as vascularized graft in a bone defect, is yet to be explored. Here, we compare the above-mentioned bone substitutes with autologous bone with the aim of selecting one for future studies of segmental mandibular repair. Six female sheep, aged 7-8 years, were implanted with 40 mm long four-chambered polyether ether ketone (PEEK) bioreactors prepared using additive manufacturing followed by plasma immersion ion implantation (PIII) to improve hydrophilicity and bioactivity. Each bioreactor was wrapped with vascularized scapular periosteum and the chambers were filled with autologous bone graft, Novabone®, BioOss®, and Zengro®, respectively. The bioreactors were implanted within a subscapular muscle pocket for either 8 weeks (two sheep), 10 weeks (two sheep), or 12 weeks (two sheep), after which they were removed and assessed by microCT and routine histology. Moderate bone formation was observed in autologous bone grafts, while low bone formation was observed in the BioOss® and Zengro® chambers. No bone formation was observed in the Novabone® chambers. Although the BioOss® and Zengro® chambers contained relatively small amounts of bone, endochondral ossification and retained hydroxyapatite suggest their potential in new bone formation in an ectopic site if a consistent supply of progenitor cells and/or growth factors can be ensured over a longer duration.
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Periosteum is a highly vascularized membrane lining the surface of bones. It plays essential roles in bone repair following injury and reconstruction following invasive surgeries. To broaden the use of periosteum, including for augmenting in vitro bone engineering and/or in vivo bone repair, we have developed an ex vivo perfusion bioreactor system to maintain the cellular viability and metabolism of surgically resected periosteal flaps. Each specimen was placed in a 3D printed bioreactor connected to a peristaltic pump designed for the optimal flow rates of tissue perfusate. Nutrients and oxygen were perfused via the periosteal arteries to mimic physiological conditions. Biochemical assays and histological staining indicate component cell viability after perfusion for almost 4 weeks. Our work provides the proof-of-concept of ex vivo periosteum perfusion for long-term tissue preservation, paving the way for innovative bone engineering approaches that use autotransplanted periosteum to enhance in vivo bone repair.
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Periósteo , Engenharia Tecidual , Ovinos , Animais , Periósteo/irrigação sanguínea , Periósteo/transplante , Retalhos Cirúrgicos , Perfusão , Reatores BiológicosRESUMO
The laboratory culture of human stem cells seeks to capture a cellular state as an in vitro surrogate of a biological system. For the results and outputs from this research to be accurate, meaningful, and durable, standards that ensure reproducibility and reliability of the data should be applied. Although such standards have been previously proposed for repositories and distribution centers, no widely accepted best practices exist for laboratory research with human pluripotent and tissue stem cells. To fill that void, the International Society for Stem Cell Research has developed a set of recommendations, including reporting criteria, for scientists in basic research laboratories. These criteria are designed to be technically and financially feasible and, when implemented, enhance the reproducibility and rigor of stem cell research.
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Pesquisa com Células-Tronco , Humanos , Reprodutibilidade dos TestesRESUMO
Tissue engineered constructs can serve as in vitro models for research and replacement of diseased or damaged tissue. As an emerging technology, 3D bioprinting enables tissue engineering through the ability to arrange biomaterials and cells in pre-ordered structures. Hydrogels, such as alginate (Alg), can be formulated as inks for 3D bioprinting. However, Alg has limited cell affinity and lacks the functional groups needed to promote cell growth. In contrast, graphene oxide (GO) can support numerous cell types and has been purported for use in regeneration of bone, neural and cardiac tissues. Here, GO was incorporated with 2% (w/w) Alg and 3% (w/w) gelatin (Gel) to improve 3D printability for extrusion-based 3D bioprinting at room temperature (RT; 25°C) and provide a 3D cellular support platform. GO was more uniformly distributed in the ink with our developed method over a wide concentration range (0.05%-0.5%, w/w) compared to previously reported GO containing bioink. Cell support was confirmed using adipose tissue derived stem cells (ADSCs) either seeded onto 3D printed GO scaffolds or encapsulated within the GO containing ink before direct 3D printing. Added GO was shown to improve cell-affinity of bioinert biomaterials by providing more bioactive moieties on the scaffold surface. 3D cell-laden or cell-seeded constructs showed improved cell viability compared to pristine (without GO) bio-ink-based scaffolds. Our findings support the application of GO for novel bio-ink formulation, with the potential to incorporate other natural and synthetic materials such as chitosan and cellulose for advanced in situ biosensing, drug-loading and release, and with the potential for electrical stimulation of cells to further augment cell function.
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Heart failure affects more than 10% of the Australian population over age 65, and the ageing population will ensure continued growth of this significant problem. There are various treatment options available, but the growing field of regenerative therapy offers promise to restore or replace tissue lost in those with either congenital or acquired cardiac defects. Stem cells have many potential properties, but they need multiple discussed qualities to succeed in this field such as ease of harvest and multiplication, and most importantly minimal ethical concerns. There are multiple cell types available and one of the challenges will be to find the most appropriate cell type for cardiac regeneration. Cardiac tissue engineering is being explored using both in vitro and in vivo techniques. In vitro methods are primarily limited in terms of the vascularisation and size of the construct. In vivo engineered constructs overcome these limitations in early models, but they are still not ready for human trials. This review aims to provide the reader with an outline of the cell-based and tissue engineering therapies currently being used and developed for cardiac regeneration, as well as some insight into the potential problems that may hamper its progress in the future.
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Cardiopatias/terapia , Miocárdio , Regeneração , Transplante de Células-Tronco , Células-Tronco , Engenharia Tecidual , Animais , HumanosRESUMO
There is a fundamental need for clinically relevant, reproducible, and standardized in vitro human neural tissue models, not least of all to study heterogenic and complex human-specific neurological (such as neuropsychiatric) disorders. Construction of three-dimensional (3D) bioprinted neural tissues from native human-derived stem cells (e.g., neural stem cells) and human pluripotent stem cells (e.g., induced pluripotent) in particular is appreciably impacting research and conceivably clinical translation. Given the ability to artificially and favorably regulate a cell's survival and behavior by manipulating its biophysical environment, careful consideration of the printing technique, supporting biomaterial and specific exogenously delivered stimuli, is both required and advantageous. By doing so, there exists an opportunity, more than ever before, to engineer advanced and precise tissue analogs that closely recapitulate the morphological and functional elements of natural tissues (healthy or diseased). Importantly, the application of electrical stimulation as a method of enhancing printed tissue development in vitro, including neuritogenesis, synaptogenesis, and cellular maturation, has the added advantage of modeling both traditional and new stimulation platforms, toward improved understanding of efficacy and innovative electroceutical development and application.
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Bioprinting is an additive manufacturing process where biomaterials-based inks are printed layer-by-layer to create three-dimensional (3D) structures that mimic natural tissues. Quality assurance for 3D bioprinting is paramount to undertaking fundamental research and preclinical and clinical product development. It forms part of quality management and is vital to reproducible and safe tissue fabrication, function, and regulatory approval for translational application. This chapter seeks to place the implementation of quality practices in 3D bioprinting front-of-mind, with emphasis on cell processing, although important to all components and procedures of the printing pipeline.
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Bioimpressão/métodos , Impressão Tridimensional , Benchmarking , Bioimpressão/normas , Biópsia , Técnicas de Cultura de Células , Terapia Baseada em Transplante de Células e Tecidos , Descoberta de Drogas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/transplante , Licenciamento , Impressão Tridimensional/normas , Controle de Qualidade , Pesquisa/economia , Pesquisa/normas , Manejo de Espécimes , Pesquisa Translacional BiomédicaRESUMO
Bioprinting cells with an electrically conductive bioink provides an opportunity to produce three-dimensional (3D) cell-laden constructs with the option of electrically stimulating cells in situ during and after tissue development. We and others have demonstrated the use of electrical stimulation (ES) to influence cell behavior and function for a more biomimetic approach to tissue engineering. Here, we detail a previously published method for 3D printing an electrically conductive bioink with human neural stem cells (hNSCs) that are subsequently differentiated. The differentiated tissue constructs comprise functional neurons and supporting neuroglia and are amenable to ES for the purposeful modulation of neural activity. Importantly, the method could be adapted to fabricate and stimulate neural and nonneural tissues from other cell types, with the potential to be applied for both research- and clinical-product development.
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Materiais Biocompatíveis , Bioimpressão , Células-Tronco Neurais , Impressão Tridimensional , Engenharia Tecidual/métodos , Cálcio/análise , Células Cultivadas , Condutividade Elétrica , Estimulação Elétrica , Corantes Fluorescentes , Humanos , Imunofenotipagem , Microscopia Confocal/métodos , Células-Tronco Neurais/transplante , Neurogênese , Neuroglia/transplante , Neurônios/transplante , Análise de Célula ÚnicaRESUMO
Bioprinting human pluripotent stem cells (PSCs) provides an opportunity to produce three-dimensional (3D) cell-laden constructs with the potential to be differentiated in vitro to all tissue types of the human body. Here, we detail a previously published method for 3D printing human induced pluripotent stem cells (iPSCs; also applicable to human embryonic stem cells) within a clinically amenable bioink (also described in Chapter 10 ) that is cross-linked to a 3D construct. The printed iPSCs continue to have self-replicating and multilineage cell induction potential in situ, and the constructs are robust and amenable to different differentiation protocols for fabricating diverse tissue types, with the potential to be applied for both research- and clinical-product development.
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Bioimpressão/métodos , Células-Tronco Pluripotentes Induzidas , Impressão Tridimensional , Engenharia Tecidual/métodos , Alginatos , Materiais Biocompatíveis , Técnicas de Cultura de Células , Sobrevivência Celular , Microambiente Celular , Células-Tronco Embrionárias Humanas , Humanos , Hidrogéis , Imunofenotipagem , Células-Tronco Pluripotentes Induzidas/citologia , Alicerces TeciduaisRESUMO
The versatile properties of graphene-based materials are enabling various tissue regeneration, towards meeting an ever increasing demand for replacement tissues due to injury through trauma and disease. In particular, an innate ability for graphene to promote osteogenic differentiation of stem cells, combined with the potential to enhance the biological activity of cells through electrical stimulation (ES) using graphene, supports its use for osteoinduction or reconstruction. In this paper, we describe a miniaturized graphene-cellulose (G-C) scaffold-based device that incorporates electroactive G-C 'paper' within a polystyrene chamber for concomitant cell culture and ES. The G-C electrodes possessed lower impedance and higher charge injection capacity than gold (Au) electrodes, with high stability. By coupling ES with previously reported properties of the G-C scaffolds, we have advanced the platform for improved adipose derived stem cell (ADSC) support and osteogenic differentiation. We anticipate using the G-C scaffold-based ES device for in vitro modelling of osteogenic induction, bone tissue engineering and in vivo bone regeneration towards new therapeutic strategies for bone injury and disease. Furthermore, the device could reasonably be used for ES and culture of other cell types and engineering other tissues.
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Tecido Adiposo/citologia , Estimulação Elétrica/instrumentação , Osteogênese/efeitos da radiação , Células-Tronco , Alicerces Teciduais/química , Técnicas de Cultura de Células/instrumentação , Células Cultivadas , Celulose/química , Desenho de Equipamento , Grafite/química , Humanos , Células-Tronco/citologia , Células-Tronco/metabolismo , Células-Tronco/efeitos da radiaçãoRESUMO
Tissue engineering, based on a combination of 3D printing, biomaterials blending and stem cell technology, offers the potential to establish customized, transplantable autologous implants using a patient's own cells. Graphene, as a two-dimensional (2D) version of carbon, has shown great potential for tissue engineering. Here, we describe a novel combination of graphene with 3D printed alginate (Alg)-based scaffolds for human adipose stem cell (ADSC) support and osteogenic induction. Alg printing was enabled through addition of gelatin (Gel) that was removed after printing, and the 3D structure was then coated with graphene oxide (GO). GO was chemically reduced with a biocompatible reductant (ascorbic acid) to provide electrical conductivity and cell affinity sites. The reduced 3D graphene oxide (RGO)/Alg scaffold has good cytocompatibility and can support human ADSC proliferation and osteogenic differentiation. Our finding supports the potential for the printed scaffold's use for in vitro engineering of bone and other tissues using ADSCs and potentially other human stem cells, as well as in vivo regenerative medicine.
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Electrical stimulation is increasingly being used to modulate human cell behaviour for biotechnological research and therapeutics. Electrically conductive polymers (CPs) such as polypyrrole (PPy) are amenable to in vitro and in vivo cell stimulation, being easy to synthesise with different counter ions (dopants) to augment biocompatibility and cell-effects. Extending our earlier work, which showed that CP-mediated electrical stimulation promotes human neural stem cell differentiation, here we report using electroactive PPy containing the anionic dopant dodecylbenzenesulfonate (DBS) to modulate the fate determination of human induced pluripotent stem cells (iPSCs). Remarkably, the stimulation without conventional chemical inducers resulted in the iPSCs differentiating to cells of the three germ lineages-endoderm, ectoderm, and mesoderm. The unstimulated iPSC controls remained undifferentiated. Phenotypic characterisation further showed a robust induction to neuronal fate with electrical stimulation, again without customary chemical inducers. Our findings add to the growing body of evidence supporting the use of electrical stimulation to augment stem cell differentiation, more specifically, pluripotent stem cell differentiation, and especially neuronal induction. Moreover, we have shown the versatility of electroactive PPy as a cell-compatible platform for advanced stem cell research and translation, including identifying novel mechanisms of fate regulation, tissue development, electroceuticals, and regenerative medicine.
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Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Polímeros/farmacologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Estimulação Elétrica/métodos , Humanos , Neurogênese/efeitos dos fármacos , Neurogênese/fisiologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Pirróis/farmacologiaRESUMO
Engineering substantia propria (or stroma of cornea) that mimics the function and anatomy of natural tissue is vital for in vitro modelling and in vivo regeneration. There are, however, few examples of bioengineered biomimetic corneal stroma. Here we describe the construction of an orthogonally oriented 3D corneal stroma model (3D-CSM) using pure electro-compacted collagen (EC). EC films comprise aligned collagen fibrils and support primary human corneal stromal cells (hCSCs). Cell-laden constructs are analogous to the anatomical structure of native human cornea. The hCSCs are guided by the topographical cues provided by the aligned collagen fibrils of the EC films. Importantly, the 3D-CSM are biodegradable, highly transparent, glucose-permeable and comprise quiescent hCSCs. Gene expression analysis indicated the presence of aligned collagen fibrils is strongly coupled to downregulation of active fibroblast/myofibroblast markers α-SMA and Thy-1, with a concomitant upregulation of the dormant keratocyte marker ALDH3. The 3D-CSM represents the first example of an optimally robust biomimetic engineered corneal stroma that is constructed from pure electro-compacted collagen for cell and tissue support. The 3D-CSM is a significant advance for synthetic corneal stroma engineering, with the potential to be used for full-thickness and functional cornea replacement, as well as informing in vivo tissue regeneration. STATEMENT OF SIGNIFICANCE: This manuscript represents the first example of a robust, transparent, glucose permeable and pure collagen-based biomimetic 3D corneal stromal model (3D-CSM) constructed from pure electro-compacted collagen. The collagen fibrils of 3D-CSM are aligned and orthogonally arranged, mimicking native human corneal stroma. The alignment of collagen fibrils correlates with the direction of current applied for electro-compaction and influences human corneal stromal cell (hCSC) orientation. Moreover, 3D-CSM constructs support a corneal keratocyte phenotype; an essential requirement for modelling healthy corneal stroma. As-prepared 3D-CSM hold great promise as corneal stromal substitutes for research and translation, with the potential to be used for full-thickness cornea replacement.
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Substância Própria , Engenharia Tecidual , Biomimética , Colágeno , Córnea , Ceratócitos da Córnea , HumanosRESUMO
Single Cell Force Spectroscopy was applied to measure the single cell de-adhesion between human neural stem cells (hNSC) and gelatin methacrylate (GelMA) hydrogel with varying modulus in the range equivalent to brain tissue. The cell de-adhesion force and energy were predominately generated via unbinding of complexes formed between RGD groups of the GelMA and cell surface integrin receptors and the de-adhesion force/energy were found to increase with decreasing modulus of the GelMA hydrogel. For the softer GelMA hydrogels (160â¯Pa and 450â¯Pa) it was proposed that a lower degree of cross-linking enables a greater number of polymer chains to bind and freely extend to increase the force and energy of the hNSC-GelMA de-adhesion. In this case, the multiple polymer chains are believed to act together in parallel like 'molecular tensors' to generate tensile forces on the bound receptors until the cell detaches. Counterintuitively for softer substrates, this type of interaction gave rise to higher force loading rates, including the appearance of high and low dynamic force regimes in de-adhesion rupture force versus loading rate analysis. For the stiffer GelMA hydrogel (900â¯Pa) it was observed that the extension and elastic restoring forces of the polymer chains contributed less to the cell de-adhesion. Due to the apparent lower extent of freely interacting chains on the stiffer GelMA hydrogel the intrinsic RGD groups are presumed to be "more fixed" to the substrate. Hence, the cell de-adhesion is suggested to be mainly governed by the discrete unbinding of integrin-RGD complexes as opposed to elastic restoring forces of polymer chains, leading to smaller piconewton rupture forces and only a single lower dynamic force regime. Intriguingly, when integrin antibodies were introduced for binding integrin α5ß1, ß1- and αv-subunits it was revealed that the cell modifies the de-adhesion force depending on the substrate stiffness. The antibody binding supressed the de-adhesion on the softer GelMA hydrogel while on the stiffer GelMA hydrogel caused an opposing reinforcement in the de-adhesion. STATEMENT OF SIGNIFICANCE: Conceptual models on cell mechanosensing have provided molecular-level insight to rationalize the effects of substrate stiffness. However most experimental studies evaluate the cell adhesion by analysing the bulk material properties. As such there is a discrepancy in the scale between the bulk properties versus the nano- and micro-scale cell interactions. Furthermore there is a paucity of experimental studies on directly measuring the molecular-level forces of cell-material interactions. Here we apply Single Cell Force Spectroscopy to directly measure the adhesion forces between human neural stem cells and gelatin-methacrylate hydrogel. We elucidate the mechanisms by which single cells bind and physically interact with hydrogels of varying stiffness. The study highlights the use of single cell analysis tools to probe molecular-level interactions at the cell-material interface which is of importance in designing material cues for regulating cell function.
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Adesão Celular/efeitos dos fármacos , Gelatina/química , Hidrogéis/química , Metacrilatos/química , Células-Tronco Neurais/metabolismo , Animais , Linhagem Celular , Módulo de Elasticidade , Humanos , CamundongosRESUMO
Human brain organoids provide opportunities to produce three-dimensional (3D) brain-like tissues for biomedical research and translational drug discovery, toxicology, and tissue replacement. Here we describe a protocol for rapid and defined induction of brain organoids from human induced pluripotent stem cells (iPSCs), using commercially available culture and differentiation media and a cheap, easy to handle and clinically approved semisynthetic hydrogel. Importantly, the methodology is uncomplicated, well-defined, and reliable for reproducible and scalable organoid generation, and amendable to principles of current good laboratory practice (cGLP), with the potential for prospective adaptation to current good manufacturing practice (cGMP) toward clinical compliance.