Cell Culture Matrix Research Articles

The Biomimetic Electrical Stimulation System Inducing Osteogenic Differentiations of BMSCs.

Electrical stimulation has been used clinically as an adjunct therapy to accelerate the healing of bone defects, and its mechanism requires further investigations. The complexity of the physiological microenvironment makes it challenging to study the effect of electrical signal on cells alone. Therefore, an artificial system mimicking cell microenvironment in vitro was developed to address this issue. In this work, a novel electrical stimulation system was constructed based on polypyrrole nanowires (ppyNWs) with a high aspect ratio. Synthesized ppyNWs formed a conductive network in the composited hydrogel which contained modified gelatin with methacrylate, providing a conductive cell culture matrix for bone marrow mesenchymal stem cells. The dual-network conductive hydrogel had improved mechanical, electrical, and hydrophilic properties. It was able to imitate the three-dimensional structure of the cell microenvironment and allowed adjustable electrical stimulations in the following system. This hydrogel was integrated with cell culture plates, platinum electrodes, copper wires, and external power sources to construct the artificial electrical stimulation system. The optimum voltage of the electrical stimulation system was determined to be 2 V, which exhibited remarkable biocompatibility. Moreover, this system had significant promotion in cell spreading, osteogenic makers, and bone-related gene expression of stem cells. RNA-seq analysis revealed that osteogenesis was correlated to Notch, BMP/Smad, and calcium signal pathways. It was proven that this biomimetic system could regulate the osteogenesis procedure, and it provided further information about how the electrical signal regulates osteogenic differentiations.

Copper ions-photo dual-crosslinked alginate hydrogel for angiogenesis and osteogenesis.

Early healing of bone defects is still a clinical challenge. Many bone-filling materials have been studied, among which photocrosslinked alginate has received significant attention due to its good biocompatibility and morphological plasticity. Although it has been confirmed that photocrosslinked alginate can be used as an extracellular matrix for 3D cell culture, it lacks osteogenesis-related biological functions. This study constructed a copper ions-photo dual-crosslinked alginate hydrogel scaffold by controlling the copper ion concentration. The scaffolds were shaped by photocrosslinking and then endowed with biological functions by copper ions crosslinking. According to in vitro research, the dual-crosslinked hydrogel increased the compressive strength and favored copper dose-dependent osteoblast differentiation and cell surface adherence of rat bone marrow mesenchymal stem cells and the expression of type I collagen (Col1), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), vascular endothelial growth factor (VEGF). In addition, hydrogel scaffolds were implanted into rat skull defects, and more angiogenesis and osteogenesis could be observed in in vivo studies. The above results show that the copper-photo-crosslinked hydrogel scaffold has excellent osseointegration properties and can potentially promote angiogenesis and early healing of bone defects, providing a reference solution for bone tissue engineering materials.

Tuning viscoelasticity and stiffness in bioprinted hydrogels for enhanced 3D cell culture: A multi-scale mechanical analysis

Bioprinted hydrogels are extensively studied to provide an artificial matrix for 3D cell culture. The success of bioprinting hydrogels relies on fine-tuning their rheology and composition to achieve shear-thinning behavior. However, a challenge arises from the limited viscoelastic and stiffness range accessible from a single hydrogel formulation. Nevertheless, hydrogel mechanical properties are recognized as essential cues influencing cell phenotype, migration, and differentiation. Thus, it is crucial to develop a system to easily modulate bioprinted hydrogels' mechanical behaviors.In this work, we modulated the viscoelastic properties and stiffness of bioprinted hydrogels composed of fibrinogen, alginate, and gelatin by tuning the crosslinking bath solution. Various concentrations of calcium ionically crosslinked alginate, while transglutaminase crosslinked gelatin. Subsequently, we characterized the mechanical behavior of our bioprinted hydrogels from the nanoscale to the macroscale. This approach enabled the production of diverse bioprinted constructs, either with similar elastic behavior but different elastic moduli or with similar elastic moduli but different viscoelastic behavior from the same hydrogel formulation.Culturing fibroblasts in the hydrogels for 33 days revealed a preference for cell growth and matrix secretion in the viscoelastic hydrogels. This work demonstrates the suitability of the method to decouple the effects of material mechanical from biochemical composition cues on 3D cultured cells.

Digital Photoinduced Topographical Microsculpting of Hydrogels

AbstractHydrogels are soft materials widely used as a substrate or matrix for cell culture to mimic the physicochemical, structural, and mechanical properties of the cell's native microenvironment. Recently, a growing body of evidence has shown that substrate topography is a key determinant of cell behavior. These studies, however, mostly rely on the use of hard elastomeric materials, particularly as introducing topography in soft hydrogels is technically challenging, labor intensive, and often does not provide a flexible design space. Here, a new approach is presented that allows robust creation of topographies on hydrogels using a maskless and contactless UV‐sculpting approach. The sculpted topographies are highly programmable: the features can be designed using any digital drawing software, while the dimensions and resolution can be easily tuned via UV exposure dose or photoinitiator concentration. Microscale topographies (2–100 µm in size) can be created on a wide range of hydrogels with diverse chemical characters and mechanical properties (e.g., stiffness). As a proof of concept, this work shows that adherent cells on softer hydrogels align better to topographical cues than on stiffer hydrogels, underlining the importance of studying cell behavior in physiologically more relevant multi‐cue environments.

Transcriptional insights from the human embryo identify laminin-511 as a suitable matrix for human trophoblast stem cell culture

The establishment of culture conditions to propagate self-renewing human trophoblast stem cells in long-term culture provides a paradigm for in vitro modelling of trophoblast. The extracellular matrix (ECM) is a critical determinant of cell identity and behaviour. Therefore, models aiming to reproduce cells in vitro should recapitulate the native cell-ECM microenvironment. Here, we mine human embryo transcriptional datasets to identify ECM components and cognate receptors expressed in the trophectoderm. Following, we identify laminin-511-E8 protein fragment as a physiologically relevant ECM capable of maintaining hTSCs in the stem cell state and retaining differentiation ability.

In situ crosslinkable multi-functional and cell-responsive alginate 3D matrix via thiol-maleimide click chemistry

In vivo, cells interact with the extracellular matrix (ECM), which provides a multitude of biophysical and biochemical signals that modulate cellular behavior. Inspired by this, we explored a new methodology to develop a more physiomimetic polysaccharide-based matrix for 3D cell culture. Maleimide-modified alginate (AlgM) derivatives were successfully synthesized using DMTMM to activate carboxylic groups. Thiol-terminated cell-adhesion peptides were tethered to the hydrogel network to promote integrin binding. Rapid and efficient in situ hydrogel formation was promoted by thiol-Michael addition “click” chemistry via maleimide reaction with thiol-flanked protease-sensitive peptides. Alginate derivatives were further ionically crosslinked by divalent ions present in the medium, which led to greater stability and allowed longer cell culture periods. By tailoring alginate's biofunctionality we improved cell-cell and cell-matrix interactions, providing an ECM-like 3D microenvironment. We were able to systematically and independently vary biochemical and biophysical parameters to elicit specific cell responses, creating custom-made 3D matrices. DMTMM-mediated maleimide incorporation is a promising approach to synthesizing AlgM derivatives that can be leveraged to produce ECM-like matrices for a broad range of applications, from in vitro tissue modeling to tissue regeneration.

Hydrogels in Cutaneous Wound Healing: Insights into Characterization, Properties, Formulation and Therapeutic Potential.

Hydrogels are polymeric materials that possess a set of characteristics meeting various requirements of an ideal wound dressing, making them promising for wound care. These features include, among others, the ability to absorb and retain large amounts of water and the capacity to closely mimic native structures, such as the extracellular matrix, facilitating various cellular processes like proliferation and differentiation. The polymers used in hydrogel formulations exhibit a broad spectrum of properties, allowing them to be classified into two main categories: natural polymers like collagen and chitosan, and synthetic polymers such as polyurethane and polyethylene glycol. This review offers a comprehensive overview and critical analysis of the key polymers that can constitute hydrogels, beginning with a brief contextualization of the polymers. It delves into their function, origin, and chemical structure, highlighting key sources of extraction and obtaining. Additionally, this review encompasses the main intrinsic properties of these polymers and their roles in the wound healing process, accompanied, whenever available, by explanations of the underlying mechanisms of action. It also addresses limitations and describes some studies on the effectiveness of isolated polymers in promoting skin regeneration and wound healing. Subsequently, we briefly discuss some application strategies of hydrogels derived from their intrinsic potential to promote the wound healing process. This can be achieved due to their role in the stimulation of angiogenesis, for example, or through the incorporation of substances like growth factors or drugs, such as antimicrobials, imparting new properties to the hydrogels. In addition to substance incorporation, the potential of hydrogels is also related to their ability to serve as a three-dimensional matrix for cell culture, whether it involves loading cells into the hydrogel or recruiting cells to the wound site, where they proliferate on the scaffold to form new tissue. The latter strategy presupposes the incorporation of biosensors into the hydrogel for real-time monitoring of wound conditions, such as temperature and pH. Future prospects are then ultimately addressed. As far as we are aware, this manuscript represents the first comprehensive approach that brings together and critically analyzes fundamental aspects of both natural and synthetic polymers constituting hydrogels in the context of cutaneous wound healing. It will serve as a foundational point for future studies, aiming to contribute to the development of an effective and environmentally friendly dressing for wounds.

Self-Degrading Multifunctional PEG-Based Hydrogels-Tailormade Substrates for Cell Culture.

The use of PEG-based hydrogels as cell culture matrix to mimic the natural extracellular matrix (ECM) has been realized using a range of well-defined, tunable, and dynamic scaffolds, although they require cell adhesion ligands such as RGDS-peptide (Arg-Gly-Asp-Ser) to promote cell adhesion. Herein the synthesis of ionic and degradable hydrogels is demonstrated for cell culture by crosslinking [PEG-SH]4 with the zwitterionic crosslinker N,N-bis(acryloxyethyl)-N-methyl-N-(3-sulfopropyl) ammonium betaine (BMSAB) and the cationic crosslinker N,N-bis(acryloxyethyl)-N,N-dimethyl-1-ammonium iodide (BDMAI). Depending on the amount of ionic crosslinker used in gel formation, the hydrogels show tunable gelation time and stiffness. At the same time, the ionic groups act as catalysts for hydrolytic degradation, thereby allowing to define a stability window. The latter could be tailored in a straightforward manner by introducing the non-degradable crosslinker tri(ethylene glycol) divinyl ether. In addition, both ionic crosslinkers favor cell attachment in comparison to the pristine PEG hydrogels. The degradation is examined by swelling behavior, rheology, and fluorescence correlation spectroscopy indicating degradation kinetics depending on diffusion of incorporated fluorescent molecules.

Studying sprouting angiogenesis under combination of oxygen gradients and co-culture of fibroblasts using microfluidic cell culture model.

Sprouting angiogenesis is an essential process for expanding vascular systems under various physiological and pathological conditions. In this paper, a microfluidic device capable of integrating a hydrogel matrix for cell culture and generating stable oxygen gradients is developed to study the sprouting angiogenesis of endothelial cells under combinations of oxygen gradients and co-culture of fibroblast cells. The endothelial cells can be cultured as a monolayer endothelium inside the device to mimic an existing blood vessel, and the hydrogel without or with fibroblast cells cultured in it provides a matrix next to the formed endothelium for three-dimensional sprouting of the endothelial cells. Oxygen gradients can be stably established inside the device for cell culture using the spatially-confined chemical reaction method. Using the device, the sprouting angiogenesis under combinations of oxygen gradients and co-culture of fibroblast cells is systematically studied. The results show that the oxygen gradient and the co-culture of fibroblast cells in the hydrogel can promote sprouting of the endothelial cells into the hydrogel matrix by altering cytokines in the culture medium and the physical properties of the hydrogel. The developed device provides a powerful in vitro model to investigate sprouting angiogenesis under various in vivo-like microenvironments.

Development of a Redox-Polymer-Based Electrochemical Glucose Biosensor Suitable for Integration in Microfluidic 3D Cell Culture Systems.

The inclusion of online, in situ biosensors in microfluidic cell cultures is important to monitor and characterize a physiologically mimicking environment. This work presents the performance of second-generation electrochemical enzymatic biosensors to detect glucose in cell culture media. Glutaraldehyde and ethylene glycol diglycidyl ether (EGDGE) were tested as cross-linkers to immobilize glucose oxidase and an osmium-modified redox polymer on the surface of carbon electrodes. Tests employing screen printed electrodes showed adequate performance in a Roswell Park Memorial Institute (RPMI-1640) media spiked with fetal bovine serum (FBS). Comparable first-generation sensors were shown to be heavily affected by complex biological media. This difference is explained in terms of the respective charge transfer mechanisms. Under the tested conditions, electron hopping between Os redox centers was less vulnerable than H2O2 diffusion to biofouling by the substances present in the cell culture matrix. By employing pencil leads as electrodes, the incorporation of these electrodes in a polydimethylsiloxane (PDMS) microfluidic channel was achieved simply and at a low cost. Under flow conditions, electrodes fabricated using EGDGE presented the best performance with a limit of detection of 0.5 mM, a linear range up to 10 mM, and a sensitivity of 4.69 μA mM-1 cm-2.

Phase Separation Microparticles as a Three-Dimensional Cell Culture System To Promote Stem Cell Expansion

Stem cell therapy is developing rapidly, but existing expansion techniques are insufficient for the use of a large number of cells. The surface chemistry and surface morphology characteristics of materials play a critical role in cellular behaviors and functions and have guiding significance for the design of biomaterials. Many studies have proven that these factors are essential to affect cell adhesion and growth. How to design a suitable biomaterial interface is the focus of recent studies. Here, the mechanosensing of human adipose-derived stem cells (hASC) on a set of materials and materials with various porosity is systematically studied. Guided by the mechanism discoveries, three-dimensional (3D) microparticles with optimized hydrophilicity and morphology are designed via liquid-liquid phase separation technology. The microparticles support scalable stem cell culture and extracellular matrix (ECM) collection, exhibiting great potential for stem cell applications.

Emerging Role of Injectable Dipeptide Hydrogels in Biomedical Applications.

Owing to their properties such as biocompatibility, tunable mechanical properties, permeability toward oxygen, nutrients, and the ability to hold a significant amount of water, hydrogels have wide applications in biomedical research. They have been engaged in drug delivery systems, 3D cell culture, imaging, and extracellular matrix (ECM) mimetics. Injectable hydrogels represent a major subset of hydrogels possessing advantages of site-specific conformation with minimal invasive techniques. It preserves the inherent properties of drug/biomolecules and is devoid of any side effects associated with surgery. Various polymeric materials utilized in developing injectable hydrogels are associated with the limitations of toxicity, immunogenicity, tedious manufacturing processes, and lack of easy synthetic tunability. Peptides are an important class of biomaterials that have interesting properties such as biocompatibility, stimuli responsiveness, shear thinning, self-healing, and biosignaling. They lack immunogenicity and toxicity. Therefore, numerous peptide-based injectable hydrogels have been explored in the past, and a few of them have reached the market. In recent years, minimalistic dipeptides have shown their ability to form stable hydrogels through cooperative noncovalent interactions. In addition to inherent properties of lengthy peptide-based injectable hydrogels, dipeptides have the unique advantages of low production cost, high synthetic accessibility, and higher stability. Given the instances of expanding significance of injectable peptide hydrogels in biomedical research and an emerging recent trend of dipeptide-based injectable hydrogels, a timely review on dipeptide-based injectable hydrogels shall highlight various aspects of this interesting class of biomaterials. This concise review that focuses on the dipeptide injectable hydrogel may stimulate the current trends of research on this class of biomaterial to translate its significance as interesting products for biomedical applications.

Poly(acrylamide) Spheroids with Tunable Elasticity for Scalable Cell Culture Applications

Microfluidics In article 2200246, Annette M. Schmidt's and her co-workers' approach toward spherical, mm-scaled PAAm hydrogel beads with adjustable, soft elastic properties as a matrix for scalable cell culture is described. The beads are produced by a continuous microfluidic approach, and after surface modification with biomarkers, their suitability as a matrix for scalable cell cultivation is successfully demonstrated with three different cell types in model experiments and in a tidal bioreactor system.

Electrospun Silk Fibroin-CNT Composite Fibers: Characterization and Application in Mediating Fibroblast Stimulation.

Electrospinning is a simple, low-cost, and highly efficient technique to generate desirable nano/microfibers from polymer solutions. Silk fibroin (SF), a biopolymer found in Bombyx mori cocoons, has attracted attention for various biomedical applications. In this study, functionalized CNT was incorporated in SF to generate biocomposite fibers by electrospinning. The electrospun (E-spun) fibers were well aligned with morphology mimicking the locally oriented ECM proteins in connective tissues. While as-spun fibers dissolved in water in just two minutes, ethanol vapor post-treatment promoted β-sheet formation leading to improved fiber stability in an aqueous environment (>14 days). The addition of a minute amount of CNT effectively improved the E-spun fiber alignment and mechanical strength while retained high biocompatibility and biodegradability. The fibers’ electrical conductivity increased by 13.7 folds and 21.8 folds, respectively, in the presence of 0.1 w% and 0.2 w% CNT in SF fibers. With aligned SF-CNT 0.1 % fibers as a cell culture matrix, we found electrical stimulation effectively activated fibroblasts from patients of pelvic organ prolapse (POP), a connective tissue disorder. The stimulation boosted the fibroblasts’ productivity of collagen III (COLIII) and collagen I (COLI) by 74 folds and 58 folds, respectively, and reduced the COLI to COLIII ratio favorable for tissue repair. The developed material and method offer a simple, direct, and effective way to remedy the dysfunctional fibroblasts of patients for personalized cell therapeutic treatment of diseases and health conditions associated with collagen disorder.

A quick pipeline for the isolation of 3D cell culture-derived extracellular vesicles.

Recent advances in cell biology research regarding extracellular vesicles have highlighted an increasing demand to obtain 3D cell culture‐derived EVs, because they are considered to more accurately represent EVs obtained in vivo. However, there is still a grave need for efficient and tunable methodologies to isolate EVs from 3D cell cultures. Using nanofibrillar cellulose (NFC) scaffold as a 3D cell culture matrix, we developed a pipeline of two different approaches for EV isolation from cancer spheroids. A batch method was created for delivering high EV yield at the end of the culture period, and a harvesting method was created to enable time‐dependent collection of EVs to combine EV profiling with spheroid development. Both these methods were easy to set up, quick to perform, and they provided a high EV yield. When compared to scaffold‐free 3D spheroid cultures on ultra‐low affinity plates, the NFC method resulted in similar EV production/cell, but the NFC method was scalable and easier to perform resulting in high EV yields. In summary, we introduce here an NFC‐based, innovative pipeline for acquiring EVs from 3D cancer spheroids, which can be tailored to support the needs of variable EV research objectives.

Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment.

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark of cardiac and musculoskeletal tissues. To study cell response to aligned microenvironments in vitro, several protocols have been developed to generate COL1 matrices with defined fiber alignment, including magnetic, mechanical, cell-based, and microfluidic methods. Of these, microfluidic approaches offer advanced capabilities such as accurate control over fluid flows and the cellular microenvironment. However, the microfluidic approaches to generate aligned COL1 matrices for advanced in vitro culture platforms have been limited to thin "mats" (<40 µm in thickness) of COL1 fibers that extend over distances less than 500 µm and are not conducive to 3D cell culture applications. Here, we present a protocol to fabricate 3D COL1 matrices (130-250 µm in thickness) with millimeter-scale regions of defined fiber alignment in a microfluidic device. This platform provides advanced cell culture capabilities to model structured tissue microenvironments by providing direct access to the micro-engineered matrix for cell culture.

The amount of membrane cholesterol required for robust cell adhesion and proliferation in serum-free condition.

Serum-containing medium is widely used to support cell attachment, stable growth and serial passaging of various cancer cell lines. However, the presence of cholesterols and lipids in serum greatly hinders the analysis of the effects of cholesterol depletion on cells in culture. In this study, we developed a defined serum-free culture condition accessible to a variety of different types of adherent cancer cells. We tested different factors that are considered essential for cell culture and various extracellular matrix for plate coating, and found cells cultured in Dulbecco's Modified Eagle's Medium (DMEM) basal media supplemented with Albumin (BSA) and insulin-transferrin-selenium-ethanolamine (ITS-X) on fibronectin-precoated plate (called as "DA-X condition") showed comparable proliferation and survival to those in a serum-containing medium. Interestingly, we observed that DA-X condition could be adapted to a wide variety of adherent cancer cell lines, which enabled the analysis of how cholesterol depletion affected cancer cells in culture. Mechanistically, we found the beneficial effects of the DA-X condition in part can be attributed to the appropriate level of membrane cholesterol, and fibronectin-mediated signaling plays an important role in the suppression of cholesterol production.

Antifouling Strategies for Electrochemical Sensors in Cell Culture Application

Electrode fouling is one of the main challenges encountered during electrochemical measurements of biological material. This process takes place at the electrode surface and negatively affects analytical parameters of the sensors[1]. The impermeable layer is formed through nonspecific adsorption and polymerization or precipitation of fouling agents, such as: proteins, lipids, phenols or neurotransmitters. There are several antifouling strategies employed to protect the surface based on the hydrophobic or zwitterionic molecules, formation of a physical barrier or chemical modification of the surface with self-assembled monolayers, polymers or hydrogels[2,3]. Antifouling material should be biocompatible, naturally inert and continuously protect the electrode in a complex environment as blood, urea, sweat or wastewater[2].Electrochemical sensors can be used for monitoring of oxygen and glucose consumption in cell cultures. In long term measurement, where sensors are permanently incorporated into cell culture matrix, oxygen and glucose sensors provide information about cell viability that can be translated to toxicity during tests of new drugs. For such systems electrodes need to withstand prolonged (at least 3-4 weeks) contact with the cell culture matrix and the surrounding medium, thus biofouling is a major issue.We have tested antifouling layers including major strategies: coating with polymers (e.g. Nafion®, phenylenediamine, polyvinyl chloride, polysilicate matrix), hydrogels (e.g. polyhydroxyethyl methacrylate), proteins (e.g. albumin), polyethylene glycol and poly-L-lactic acid. Electrodes were first coated with syringaldazine, which is easily adsorbed on carbon surfaces and provides facile to interpret response towards pH changes[4]. It is also very stable in buffer solutions but quickly desorbs in more complex media, thus making it an excellent probe for measurement of beneficial impact of antifouling layers. Tests were first conducted on macro- glassy carbon electrodes and later on low-cost pencil based sensors (250 µm diameter) as it was shown that some layers are less stable due to the big size of the electrode surface. It was revelated that polysilicate matrix, poly-L-lactic acid, polyhydroxyethyl methacrylate are suitable candidates for antifouling layers for electrochemical sensors. All three were tested as sensors in HELA cell culture, assessing cell growth on the layers and around the electrodes. Stability of the layers was evaluated during three weeks of cell culture.

A Biostable l-DNA Hydrogel with Improved Stability for Biomedical Applications.

DNA hydrogels have attracted increasing attention owing to their excellent permeability and high mechanical strength, together with thixotropy, versatile programmability and good biocompatibility. However, the moderate biostability and immune stimulation of DNA have arisen as big concerns for future potential clinical applications. Herein, we report the self-assembly of a novel l-DNA hydrogel, which inherited the extraordinary physical properties of a d-DNA hydrogel. With the mirror-isomer deoxyribose, this hydrogel exhibited improved biostability, withstanding fetal bovine serum (FBS) for at least 1 month without evident decay of its mechanical properties. The low inflammatory response of the l-DNA hydrogel has been verified both in vitro and in vivo. Hence, this l-DNA hydrogel with outstanding biostability and biocompatibility can be anticipated to serve as an ideal 3D cell-culture matrix and implanted bio-scaffold for long-term biomedical applications.

Development of an efficient single-cell cloning and expansion strategy for genome edited induced pluripotent stem cells.

Disease-specific human induced pluripotent stem cells (hiPSCs) can be generated directly from individuals with known disease characteristics or alternatively be modified using genome editing approaches to introduce disease causing genetic mutations to study the biological response of those mutations. The genome editing procedure in hiPSCs is still inefficient, particularly when it comes to homology directed repair (HDR) of genetic mutations or targeted transgene insertion in the genome and single cell cloning of edited cells. In addition, genome editing processes also involve additional cellular stresses such as poor cell viability and genetic stability of hiPSCs. Therefore, efficient workflows are desired to increase genome editing application to hiPSC disease models and therapeutic applications. To this end, we demonstrate an efficient workflow for feeder-free single cell clone generation and expansion in both CRISPR-mediated knock-out (KO) and knock-in (KI) hiPSC lines. Using StemFlex medium and CloneR supplement in conjunction with Matrigel cell culture matrix, we show that cell viability and expansion during single-cell cloning in edited and unedited cells is significantly enhanced. Keeping all factors into account, we have successfully achieved hiPSC single-cell survival and cloning in both edited and unedited cells with rates as maximum as 70% in less than 2weeks. This simplified and efficient workflow will allow for a new level of sophistication in generating hiPSC-based disease models to promote rapid advancement in basic research and also the development of novel cellular therapeutics.