Cell Culture Surfaces Research Articles

Gold cleaning methods for preparation of cell culture surfaces for self-assembled monolayers of zwitterionic oligopeptides

Self-assembled monolayers (SAMs) have been used to elucidate interactions between cells and material surface chemistry. Gold surfaces modified with oligopeptide SAMs exhibit several unique characteristics, such as cell-repulsive surfaces, micropatterns of cell adhesion and non-adhesion regions for control over cell microenvironments, and dynamic release of cells upon external stimuli under culture conditions. However, basic procedures for the preparation of oligopeptide SAMs, including appropriate cleaning methods of the gold surface before modification, have not been fully established. Because gold surfaces are readily contaminated with organic compounds in the air, cleaning methods may be critical for SAM formation. In this study, we examined the effects of four gold cleaning methods: dilute aqua regia, an ozone water, atmospheric plasma, and UV irradiation. Among the methods, UV irradiation most significantly improved the formation of oligopeptide SAMs in terms of repulsion of cells on the surfaces. We fabricated an apparatus with a UV light source, a rotation table, and HEPA filter, to treat a number of gold substrates simultaneously. Furthermore, UV-cleaned gold substrates were capable of detaching cell sheets without serious cell injury. This may potentially provide a stable and robust approach to oligopeptide SAM-based experiments for biomedical studies.

Long-Term Maintenance of Human Pluripotent Stem Cells on cRGDfK-Presenting Synthetic Surfaces

Synthetic human pluripotent stem cell (hPSC) culture surfaces with defined physical and chemical properties will facilitate improved research and therapeutic applications of hPSCs. In this study, synthetic surfaces for hPSC culture in E8 medium were produced for screening by modifying two polymer brush coatings [poly(acrylamide-co-acrylic acid) (PAAA) and poly(acrylamide-co-propargyl acrylamide) (PAPA)] to present single peptides. Adhesion of hPSC colonies was more consistently observed on surfaces modified with cRGDfK compared to surfaces modified with other peptide sequences tested. PAPA-coated polystyrene flasks with coupled cRGDfK (cRGDfK-PAPA) were then used for long-term studies of three hPSC lines (H9, hiPS-NHF1.3, Genea-02). Cell lines maintained for ten passages on cRGDfK-PAPA were assessed for colony morphology, proliferation rate, maintenance of OCT4 expression, cell viability at harvest, teratoma formation potential, and global gene expression as assessed by the PluriTest™ assay. cRGDfK-PAPA and control cultures maintained on Geltrex™ produced comparable results in most assays. No karyotypic abnormalities were detected in cultures maintained on cRGDfK-PAPA, while abnormalities were detected in cultures maintained on Geltrex™, StemAdhere™ or Synthemax™. This is the first report of long term maintenance of hPSC cultures on the scalable, stable, and cost-effective cRGDfK-PAPA coating.

Design of Temperature-Responsive Cell Culture Surfaces for Cell Sheet-Based Regenerative Therapy and 3D Tissue Fabrication.

This chapter describes the concept of "cell sheet engineering" for the creation of transplantable cellular tissues and organs. In contrast to scaffold-based tissue engineering, cell sheet engineering facilitates the reconstruction of scaffold-free, cell-dense tissues. Cell sheets were harvested by changing the temperature of thermoresponsive cell culture surfaces modified with poly(N-isopropylacrylamide) (PIPAAm) with a thickness on the nanometer scale. The transplantation of 2D cell sheet tissues has been used in clinical settings. Although 3D tissues were formed simply by layering 2D cell sheets, issues related to vascularization within 3D tissues and the large-scale production of cells must be addressed to create thick and large 3D tissues and organs.

Temperature-Responsive Cell Culture Surface for Cell-Sheet Tissue Engineering and Its Design to Express Temperature-Dependent Cell Attachment/Detachment Character

Temperature-Responsive Cell Culture Surface for Cell-Sheet Tissue Engineering and Its Design to Express Temperature-Dependent Cell Attachment/Detachment Character

Surface modification of PHBV nanofiber mats for rapid cell cultivation and harvesting

To maintain the original function of a specific tissue for therapeutic tissue engineering, an advanced cell culture surface for repeat cell proliferation is necessary. We designed a novel cell proliferation and rapid harvesting surface by combining nonwoven nanofiber mat and a thermo-responsive polymer. Nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) mats were fabricated by the electrospinning technique. A poly(N-isopropylacrylamide) (PNIPAM) thermo-responsive layer was grafted on the PHBV nanofiber mat by electron beam irradiation. The average diameter of the PNIPAM-grafted PHBV nanofibers was determined by SEM. ATR-FTIR and ESCA were used to confirm the grafting of PNIPAM onto the PHBV nanofiber surface. Water contact angles on the mats were measured in response to temperature changes. Human adipose-derived stem cells (ADSCs) were cultured on the PNIPAM-grafted PHBV nanofiber mat to investigate cell proliferation, harvesting, and functionality during repeat subculture. Detached ADSCs from each surface by low temperature treatment and trypsin-EDTA were compared by a fluorescence-activated cell sorter (FACS) using expression of stem cell membrane-specific markers such as CD-13 PE, CD-29 PE, and CD-90 FITC. The mass cultivation and intact harvesting of stem cells by low temperature treatment using a thermo-responsive PHBV nanofiber mat is a promising technique for use in regenerative medicine and stem cell therapy.

Microfluidic device-assisted etching of p-HEMA for cell or protein patterning.

The construction of biomaterials with which to limit the growth of cells or to limit the adsorption of proteins is essential for understanding biological phenomena. Here, we describe a novel method to simply and easily create thin layers of poly (2-hydroxyethyl methacrylate) (p-HEMA) for protein and cellular patterning via etching with ethanol and microfluidic devices. First, a cell culture surface or glass coverslip is coated with p-HEMA. Next, a polydimethylsiloxane (PDMS) microfluidic is placed onto the p-HEMA surface, and ethanol is aspirated through the device. The PDMS device is removed, and the p-HEMA surface is ready for protein adsorption or cell plating. This method allows for the fabrication of 0.3 µm thin layers of p-HEMA, which can be etched to 10 µm wide channels. Furthermore, it creates regions of differential protein adhesion, as shown by Coomassie staining and fluorescent labeling, and cell adhesion, as demonstrated by C2C12 myoblast growth. This method is simple, versatile, and allows biologists and bioengineers to manipulate regions for cell culture adhesion and growth. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:243-248, 2018.

Growth-Factor-Releasing Polyelectrolyte Multilayer Films to Control the Cell Culture Environment.

Polyelectrolyte multilayers (PEMs) are of great interest as cell culture surfaces because of their ability to modify topography and surface energy and release biologically relevant molecules such as growth factors. In this work, fibroblast growth factor 2 (FGF2) was adsorbed directly onto polystyrene, plasma-treated polystyrene, and glass surfaces with a poly(methacrylic acid) and poly-l-histidine PEM assembled above it. Up to 14 ng/cm2 of FGF2 could be released from plasma-treated polystyrene surfaces over the course of 7 days with an FGF2 solution concentration of 100 μg/mL applied during the adsorption process. This release rate could be modulated by adjusting the adsorption concentration, decreasing to as low as 2 ng/cm2 total release over 7 days using a 12.5 μg/mL FGF2 solution. The surface energy and roughness could also be regulated using the adsorbed PEM. These properties were found to be substrate- and first-layer-dependent, supporting current theories of PEM assembly. When released, FGF2 from the PEMs was found to significantly enhance fibroblast proliferation as compared to culture conditions without FGF2. The results showed that growth factor release profiles and surface properties are easily controllable through modification of the PEM assembly steps and that these strategies can be effectively applied to common cell culture surfaces to control the cell fate.

An electrochemical study of microporous track-etched membrane permeability and the effect of surface protein layers

Microporous track-etched membranes serve as important permeable growth surfaces for cell culture where diffusive solute transport is needed across two growth compartments. This study has established effective solute diffusion coefficients for four probe micro-solutes: hydrogen peroxide, pyrocatechol, acetaminophen and ascorbic acid across three track-etched membranes formulated, respectively, from polycarbonate and polyethylene terephthalate. Chronoamperometry and cyclic voltammetry were used for the diffusion measurements. These showed substantially reduced intra-pore diffusion in relation to available pore area. Diffusion coefficients ranging from 1.43×10−10 to 3.17×10−7cm2s−1 were demonstrated. This strongly suggests that water organisation in micro-pores is not equivalent to that of bulk water. Superimposed protein layers of Type I and IV collagen, Type I collagen-fibronectin, Type I collagen-heparin, and Type I collagen-chondroitin sulphate increased diffusional resistance, but with disproportional retardation of ascorbate diffusion due to charge repulsion at collagen-heparin and collagen-chondroitin sulphate combinations. Diffusive resistance at natural tendon and cartilage was considerably smaller; diffusion coefficients ranged from 8.33×10−6 to 1.09×10−8cm2s−1.

Development of an In Vitro Exposure System for Live Visualization of the Health Impacts of Oily Marine Aerosol on the Human Respiratory System

Aerosolization of oily water droplets has recently been recognized as a potential respiratory health threat to oil spill cleanup workers, communities near spills, and marine mammals in oil-polluted waters. These sub-micron to millimeter scale droplets may be aerosolized by bursting bubbles, breaking waves, and splashing raindrops. Furthermore, dispersant applied to oil slicks also may become aerosolized as oil-water-dispersant emulsion droplets and subsequently inhaled, with unknown health consequences. With the goal of investigating the effects of inhaled oily marine aerosol on human lung health, we present the design of a novel in vitro bioreactor which mimics the conditions and exposures that human lungs might experience in the field. The bioreactor provides the ability to expose human lung cell cultures to laboratory-produced, well-characterized and chemically analyzed oily marine aerosols. A major advance over similar systems currently used to study the effects of smoking is the incorporation of optical access to allow visualization of the cells throughout exposure. In the bioreactor, differentiated, primary human bronchial epithelial cell cultures reside on membranes at the air-liquid interface between the flow-through test atmosphere and a temperature-controlled bath of culture media, thereby simulating the human lung. Oily marine aerosol is produced by a 1-Jet Collison Nebulizer (Mesa Labs Inc.) to match realistic concentrations produced and measured in a wave tank and is sampled via scanning mobility particle sizer (TSI Inc) to characterize its size distribution. The aerosol-laden air is humidified and injected at a controlled flow rate of ~1 ml/s into a module containing the cell culture, allowing particles to deposit on the cells. The module has sealed glass windows to allow optical access. An optical setup incorporating a 20× long-distance objective, 1× tube lens, and camera is used to visualize the cells over time. Preliminary testing involves determining the effectiveness of deposition of oily marine aerosol droplets at various concentrations onto the cell culture surface. Phase contrast microscopy is used to examine contact between cells as a determinant of monolayer integrity. Immunofluorescence of live cells is used with FITC- or mCherry-labelled cytoskeletal and cell-cell adhesion proteins, such as actin and E-cadherin, to determine underlying mechanisms disrupting the monolayer. A system such as this allowing for live visualization of cells during the exposure currently does not exist and will provide significant understanding of how changes within the epithelium may disrupt tissue integrity in response to inhalation of oily marine aerosol.

Acoustic tweezing cytometry enhances osteogenesis of human mesenchymal stem cells through cytoskeletal contractility and YAP activation.

Human mesenchymal stem cells (hMSCs) have great potential for cell-based therapies for treating degenerative bone diseases. It is known that mechanical cues in the cell microenvironment play an important role in regulating osteogenic (bone) differentiation of hMSCs. However, mechanoregulation of lineage commitment of hMSCs in conventional two-dimensional (2D) monocultures or bioengineered three-dimensional (3D) tissue constructs remains suboptimal due to complex biomaterial design criteria for hMSC culture. In this study, we demonstrate the feasibility of a novel cell mechanics and mechanobiology tool, acoustic tweezing cytometry (ATC), for mechanical stimulation of hMSCs. ATC utilizes ultrasound (US) pulses to actuate functionalized lipid microbubbles (MBs) which are covalently attached to hMSCs via integrin binding to exert forces to the cells. ATC stimulation increases cytoskeletal contractility of hMSCs regardless of the cell area. Furthermore, ATC application rescues osteogenic differentiation of hMSCs in culture conditions that are intrinsically repressive for hMSC osteogenesis (e.g., soft cell culture surfaces). ATC application activates transcriptional regulator YAP to enhance hMSC osteogenesis. Our data further show that F-actin, myosin II, and RhoA/ROCK signaling functions upstream of YAP activity in mediating ATC-stimulated hMSC osteogenesis. With the capability of applying controlled dynamic mechanical stimuli to cells, ATC provides a powerful tool for mechanoregulation of stem cell behaviors in tissue engineering and regenerative medicine applications.

Native and solubilized decellularized extracellular matrix: A critical assessment of their potential for improving the expansion of mesenchymal stem cells

Current mesenchymal stem cell (MSC) culture methods result in premature cellular senescence or loss of differentiation potential. This creates a major bottleneck in their clinical application, as prolonged expansion is necessary to achieve clinically relevant numbers of cells. Recently, decellularized extracellular matrix (dECM) produced by primary MSC has emerged as an attractive substrate for the improved expansion of MSC; cells cultured on these surfaces retain their desired stem cell characteristics for prolonged times during culture. This review article describes the inception and development of this dECM-based technology, points out existing challenges that must be addressed, and suggests future directions of research. To our knowledge, this is the first review written on the use of dECM for improved mesenchymal stem cell expansion.

Human umbilical cord derived matrix: Ascaffold suitable for tissue engineering application.

Human tissue derived natural extracellular matrix (ECM) has great potential in tissue engineering. We sought to isolate extracellular matrix derived from human umbilical cord and test its potential in tissue engineering. An enzymatic method was applied to isolate and solubilized complete human umbilical cord derived matrix (hUCM). The obtained solution was analyzed for growth factors, collagen and residual DNA contents, then used to coat 2D and 3D surfaces for cell culture application. The hUCM was successfully isolated with trypsin digestion to acquire a solution containing various growth factors and collagen but no residual DNA. This hUCM solution can form a coating on 2D and 3D substrates suitable cell culture. We developed a new matrix derived from human source that can be further used in tissue engineering.

Extracellular matrix mimicking scaffold promotes osteogenic stem cell differentiation: A new approach inosteoporosis research.

Osteoporosis is a common metabolic disease, with mesenchymal stem cells discussed to play an important role in its pathomechanism. For in vitro osteoporosis studies, selection of adequate culture conditions is mandatory so as to preserve cell properties as far as possible. A suitable cell culture surface would ideally provide reproducible experimental conditions by resembling those in-vivo. Generating an improved growth surface for osteogenic differentiation of human bone marrow derived mesenchymal stem cells (hBMSCs). We modified electrospun gelatine meshes with hydroxyapatite nanopowder. The potential beneficial impact of the ensuing culture conditions were evaluated by cultivating and comparing the growth of cells from osteoporotic and non-osteoporotic donors on either hydroxyapatite-gelatine (HA) meshes, pure gelatine meshes, or 2D standard tissue culture surfaces. After 21 days of differentiation, cells grown on pure or HA-gelatine meshes showed significantly higher mineralization levels compared to cells cultured in standard conditions. The amount of mineralization varied considerably in hBMSC cultures of individual patients but showed no significant difference between stem cells obtained from osteoporotic or non-osteoporotic donors. Overall, these results indicate that the use of HA-gelatine meshes as growth surfaces may serve as a valuable tool for cultivation and differentiation of mesenchymal stem cells along the osteogenic lineage, facilitating future research on osteoporosis and related issues.

Tunable Volumetric Density and Porous Structure of Spherical Poly-ε-caprolactone Microcarriers, as Applied in Human Mesenchymal Stem Cell Expansion.

Polymeric microspheres may serve as microcarrier (MC) matrices, for the expansion of anchorage-dependent stem cells. They require surface properties that promote both initial cell adhesion and the subsequent spreading of cells, which is a prerequisite for successful expansion. When implemented in a three-dimensional culture environment, under agitation, their suspension under low shear rates depends on the MCs having a modest negative buoyancy, with a density of 1.02-1.05 g/cm3. Bioresorbable poly-ε-caprolactone (PCL), with a density of 1.14 g/cm3, requires a reduction in volumetric density, for the microspheres to achieve high cell viability and yields. Uniform-sized droplets, from solutions of PCL dissolved in dichloromethane (DCM), were generated by coaxial microfluidic geometry. Subsequent exposure to ethanol rapidly extracted the DCM solvent, solidifying the droplets and yielding monodisperse microspheres with a porous structure, which was demonstrated to have tunable porosity and a hollow inner core. The variation in process parameters, including the molecular weight of PCL, its concentration in DCM, and the ethanol concentration, served to effectively alter the diffusion flux between ethanol and DCM, resulting in a broad spectrum of volumetric densities of 1.04-1.11 g/cm3. The solidified microspheres are generally covered by a smooth thin skin, which provides a uniform cell culture surface and masks their internal porous structure. When coated with a cationic polyelectrolyte and extracellular matrix protein, monodisperse microspheres with a diameter of approximately 150 μm and densities ranging from 1.05-1.11 g/cm3 are capable of supporting the expansion of human mesenchymal stem cells (hMSCs). Validation of hMSC expansion was carried out with a positive control of commercial Cytodex 3 MCs and a negative control of uncoated low-density PCL MCs. Static culture conditions generated more than 70% cell attachment and similar yields of sixfold cell expansion on all coated MCs, with poor cell attachment and growth on the negative control. Under agitation, coated porous microspheres, with a low density of 1.05 g/cm3, achieved robust cell attachment and resulted in high cell yields of ninefold cell expansion, comparable with those generated by commercial Cytodex 3 MCs.

Understanding the Interactions of Peptides and Proteins that are Commonly used in Biological Media on Polystyrene Surfaces

Polystyrene(PS) is a commonly used material for designing cell culture surfaces like assay plates, petridishes, T flasks to name a few. These surfaces when used for the culture of adherent cells will modulate cell growth and function depending of the quality of adhesion provided. To enable biological activity on polystyrene, surfaces can be modified by different treatments which result in an increase in the amount of certain surface chemical species like oxygen. To understand the differences between treated and untreated polystyrene surfaces, we examine in this study the interaction between two commonly occurring biological entities in these applications i) Albumin which is a common component of cell culture media and is used to sustain the growth and activity of biological cells and ii) commonly occurring peptide motifs that are a typical part of the ECM proteins which allow the cells to interact with the surface through these binding motifs.

Surface geometry of poly(ether imide) boosts mouse pluripotent stem cell spontaneous cardiomyogenesis via modulating the embryoid body formation process.

The permanent loss of cardiomyocytes may lead to the irreversible damage of myocardium in cardiovascular diseases. The induced pluripotent stem cells (iPSCs) with the capacity of differentiation into a variety of cell types including cardiomyocytes showed high potential for efficient heart regeneration. The iPSCs and iPSC-derived embryoid bodies (EBs) as well as the differentiated cardiomyocytes are highly sensitive to the biophysical cues of their microenvironment, and accordingly their behavior and function can be largely modulated by microstructure of the cell culture surface. In this study, we investigated the regulatory effect of microscale roughness on both cardiomyogenesis and secretion of EBs using poly(ether imide) (PEI) cell culture inserts with different levels of bottom roughness (R0: flat surface; R1: rough surface, Rq ∼ 4 μm; R2: rough surface, Rq ∼ 23 μm). The proliferation rate and cardiomyogenesis of EBs increased with the increase of surface roughness. The EB secretome derived from R2 surface remarkably enhanced the in vitro new vessel formation of endothelial cells, as compared to those from R0 and R1. These findings highlight the potential to improve the iPSC/EB-based restoration of cardiovascular function via microstructured biomaterials.

Abstract B02: Open multi-microwell array for the study of paracrine signaling in tumors

Abstract The development and progression of tumors are modulated by the interaction among multiple cell types present at the tumor adjacent tissue. Few in vitro cell culture platforms allow one to test multi-cellular interactions and those suitable require high numbers of cells limiting studies to cell lines and excluding heterogeneous patient samples, and fail to provide tunable discrete adjacent compartments for the retrieval of independent cell population readouts. Mixing of cells in adjacent compartments and cell sorting after mixed culture represent a costly and time consuming procedure that often results is lost, reduction and/or damage of the recovered sample. To overcome some of these limitations we have developed an open multi-microwell array that enables co-culture of up to 4 cell populations in discrete adjacent wells using either 2- or 3-dimension culture conditions. The circular microwells are made of poly-dimethyl siloxane (PDMS) and placed on top of glass or tissue culture plastic. The open-well format allows cells and culture media to be manually loaded and replaced using a pipette independently of passive pumping method, and without the need of vacuum-assisted filling or syringe pumps. The microwell array is aligned to a 96well plate format for automated fluorescent readouts. The cell culture surface area of each microwell is 5.7mm2 (5ul) and can accommodate 5,000-10,000 cells but the design of each microwell can be scale-down to accommodate smaller cell samples. The adjacent microwells are contained within an outer-ring of 8mm diameter and 750um height. After cell seeding, adjacent compartments can be connected by dispensing a volume of 30-40ul inside the outer ring. A model of tumor-mesenchyme paracrine Hedgehog signaling was developed and characterized to show the feasibility of this array for multi-cell soluble factor signaling studies. Activation of Hedgehog (Hh) signaling in the mesenchyme promotes proliferation of tumor cells via secreted factors. As a source of mesenchymal cell sub-types we evaluated TGF-b treated fibroblasts and tumor epithelial cells that undergo epithelial-mesenchymal transition. Myofibroblast phenotype was confirmed based on overexpression of smooth muscle actin, vimentin and fibroblast activating protein, and reduce levels of caveolin-1. Active Hh signaling and pharmacological inhibition with cyclopamine were confirmed by up- and down- regulation of canonical Hh target genes Gli1 and Patch1. Active hedgehog signaling and cell viability was maintained over 5-7days 2-dimensional and collagen type I embedded mesenchymal cells. Breast-derived human epithelial and mesenchymal cells co-cultured in microwells and transwells showed similar increase in tumor cell proliferation rates but with a reduction of 80% or more in the total cells needed compared to transwells. Flow cytometer read-outs of proliferation and tumor cell distribution of CD44/CD24 cell surface receptors were similar among transwells and microwells. Adjacent culture of mouse derived fibroblasts (NIH-3T3) and human tumor cells showed recovery of genomic sample without cell-population mixing using primers for specific for human and mouse GAPDH. Doubling of the number of microwells with mesenchymal cells modulated the observed proliferative effect in tumor cells suggesting that mesenchymal cell ratios modulate tumor response to paracrine Hh signaling. This observation could not be replicated in macro-scale platforms as active Hh signaling in the mesenchyme is achieved via cell growth arrest using a combination of low serum and cell confluence. The open multi-microwell array represents a new user-friendly multi-culture microscale array with optimized capabilities for the study of paracrine signaling among discrete cell populations compared to macroscale culture technology. Citation Format: Karla P. Ramos, Fernando Boria, Maribella Domenech. Open multi-microwell array for the study of paracrine signaling in tumors. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B02.

Scaling-Up Techniques for the Nanofabrication of Cell Culture Substrates via Two-Photon Polymerization for Industrial-Scale Expansion of Stem Cells.

Stem-cell-based therapies require a high number (106–109) of cells, therefore in vitro expansion is needed because of the initially low amount of stem cells obtainable from human tissues. Standard protocols for stem cell expansion are currently based on chemically-defined culture media and animal-derived feeder-cell layers, which expose cells to additives and to xenogeneic compounds, resulting in potential issues when used in clinics. The two-photon laser polymerization technique enables three-dimensional micro-structures to be fabricated, which we named synthetic nichoids. Here we review our activity on the technological improvements in manufacturing biomimetic synthetic nichoids and, in particular on the optimization of the laser-material interaction to increase the patterned area and the percentage of cell culture surface covered by such synthetic nichoids, from a low initial value of 10% up to 88% with an optimized micromachining time. These results establish two-photon laser polymerization as a promising tool to fabricate substrates for stem cell expansion, without any chemical supplement and in feeder-free conditions for potential therapeutic uses.

The Effects of Carbon and Gold Ion Implanted Surfaces on Neuronal Stem Cells’ Functions

Biomaterials have been used in medicine for decades to improve the functions of tissues and organs. They are also used as prosthesis and implants which are designed to substitute functions of a lacking organ or tissue. Carbon (C) and Gold (Au) were particularly chosen due to their biocompatibility and applied as implants for decades. Carbon and gold were great ion sources for medical applications, as well. In this study, polystyrene dishes were modified using gold and carbon ions via ion implantation technique. Using this surface modification method, it was aimed to improve surface characteristics and achieve a bioactive surface for neural stem cells. Even though the integration of stem cells was promising, neural stem cell studies still have many milestones to reach. Neuro-regeneration was the most desired function for people who suffer from neural system diseases. Changing surface characteristics of scaffolds was a way to promote regeneration and ion implantation was one of the methods to modify surface properties which play a huge role in enhancing the proliferation and integration of cells. In this study, it was observed that the ion implantation stimulated the neural proliferation and the implantation of different ions on cell culture surfaces was essential to determine the effects of this technique on adhesion, proliferation, differentiation and apoptosis properties of cells in details.

Upgrading well plates using open microfluidic patterning.

Cellular communication between multiple cell types is a ubiquitous process that is responsible for vital physiological responses observed in vivo (e.g., immune response, organ function). Many in vitro coculture strategies have been developed, both in traditional culture and microscale systems, and have shown the potential to recreate some of the physiological behaviors of organs or groups of cells. A fundamental limitation of current systems is the difficulty of reconciling the additional engineering requirements for creating soluble factor signaling systems (e.g., segregated cell culture) with the use of well-characterized materials and platforms that have demonstrated successful results and biocompatibility in assays. We present a new open-microfluidic platform, the Monorail Device, that is placed in any existing well plate or Petri dish and enables patterning of segregated coculture regions, thereby allowing the direct upgrade of monoculture experiments into multiculture assays. Our platform patterns biocompatible hydrogel walls via microfluidic spontaneous capillary flow (SCF) along a rail insert set inside commercially available cultureware, creating customized pipette-accessible cell culture chambers that require fewer cells than standard macroscale culture. Importantly, the device allows the use of native surfaces without additional modification or treatments, while creating permeable dividers for the diffusion of soluble factors. Additionally, the ease of patterning afforded by our platform makes reconfiguration of the culture region as simple as changing the rail insert. We demonstrate the ability of the device to pattern flows on a variety of cell culture surfaces and create hydrogel walls in complex and precise shapes. We characterize the physical parameters that enable a reproducible SCF-driven flow and highlight specialized design features that increase the ease of use of the device and control of the open microfluidic flow. Further, we present the performance of our platform according to useful coculture criteria, including permeability and integrity of our hydrogel walls and surface-sensitive cell culture. Lastly, we show the potential of this type of platform to create modular multikingdom culture systems that can be used to study soluble factor signaling between mammalian cells, bacteria, and fungi, as well as the potential for adaptation of this technology by researchers across multiple fields.