Polystyrene (PS) has brought in vitro cell culture from its humble beginnings to the modern era, propelling dozens of research fields along the way. This review discusses the development of the material, fabrication, and treatment approaches to create the culture material. However, native PS surfaces poorly facilitate cell adhesion and growth in vitro. To overcome this, liquid surface deposition, energetic plasma activation, and emerging functionalization methods transform the surface chemistry. This review seeks to highlight the many potential applications of the first widely accepted polymer growth surface. Although the majority of in vitro research occurs on two-dimensional surfaces, the importance of three-dimensional (3D) culture models cannot be overlooked. The methods to transition PS to specialized 3D culture surfaces are also reviewed. Specifically, casting, electrospinning, 3D printing, and microcarrier approaches to shift PS to a 3D culture surface are highlighted. The breadth of applications of the material makes it impossible to highlight every use, but the aim remains to demonstrate the versatility and potential as both a general and custom cell culture surface. The review concludes with emerging scaffolding approaches and, based on the findings, presents our insights on the future steps for PS as a tissue culture platform.

Although the process of chemosensing by individual cells is intrisically stochastic, multicellular organisms exhibit highly regulated responses to external stimulations. Two key elements to understand the deterministic features of chemosensing are intercellular communications and the role of pacemaker cells. To characterize the collective behavior induced by these two factors, we study the spatial-temporal calcium dynamics of fibroblast cells in response to ATP stimulation. We find that closely packed cell colonies exhibit faster, more synchronized, and highly correlated responses compared to isolated cells. In addition, we demonstrate for chemosensing the existence of pacemaker cells and how the presence of gap junctions impact the first step of the collective response. By further comparing these results with the calcium dynamics of cells embedded in thin hydrogel films, where intercellular communication is only possible via diffusing molecules, we conclude that gap junctions are required for synchronized and highly correlated responses among cells in high density colonies. In addition, in high density cell colonies, both communication channels lead to calcium oscillations following the stimulation by external ATP. While the calcium oscillations associated with cells directly exposed to external flows were transient, the oscillations of hydrogel trapped cells can persist with a fundamental frequency and higher harmonics. Our observations and measurements highlight the crucial role of intercellular signaling for generating regulated spatial and temporal dynamics in cell colonies and tissues. emergent behavior | cell signaling C ells constantly sense their local chemical environment and make decisions based upon the information received. This process, known as chemosensing, is intrinsically stochastic. Not only do the chemical perturbations fluctuate in space and time, but even for uniform stimulations, the responses vary significantly from cell to cell (1). Despite this variability, multicellular organisms are capable of performing highly regulated, consistent responses, which are crucial to maintain the normal functionality of complex life systems (2).Achieving coordinated collective responses requires individual cells to exchange information with each other within the population. For example, in the process of quorum-sensing, bacteria synthesize and secrete signaling molecules into the extracellular space. Once the concentration of the molecules, which encodes information of cell density, reaches a threshold value, bacteria transform to a different state thus generating collective behavior (3). Similarly, when a population of the social amoebae Dictyostelium discoideum is stimulated by external cAMP (cyclic adenosine 3,5-monophosphate), more cAMP is released by each cell into the extracellular space. Because Dictyostelium undergoes quiescent to oscillatory transitions as a function of external cAMP concentration, the collective response of Dictyostelium to cAMP can be dramatically different from the response o...

Cells constantly sense their chemical and mechanical environments. We study the effect of mechanics on the ATP-induced collective calcium response of fibroblast cells in experiments that mimic various tissue environments. We find that closely packed two-dimensional cell cultures on a soft polyacrylamide gel (Young's modulus E = 690 Pa) contain more cells exhibiting calcium oscillations than cultures on a rigid substrate (E = 36 000 Pa). Calcium responses of cells on soft substrates show a slower decay of calcium level relative to those on rigid substrates. Actin enhancement and disruption experiments for the cell cultures allow us to conclude that actin filaments determine the collective Ca(2+) oscillatory behaviour in the culture. Inhibition of gap junctions results in a decrease of the oscillation period and reduced correlation of calcium responses, which suggests additional complexity of signalling upon cell-cell contact. Moreover, the frequency of calcium oscillations is independent of the rigidity of the substrate but depends on ATP concentration. We compare our results with those from similar experiments on individual cells. Overall, our observations show that collective chemical signalling in cell cultures via calcium depends critically on the mechanical environment.

In the past few decades, the field of tissue engineering combined with rapid prototyping (RP) techniques has been successful in creating biological substitutes that mimic tissues. Its applications in regenerative medicine have drawn efforts in research from various scientific fields, diagnostics, and clinical translation to therapies. While some areas of therapeutics are well developed, such as skin replacement, many others such as cartilage repair can still greatly benefit from tissue engineering and RP due to the low success and/or inefficiency of current existing, often surgical treatments. Through fabrication of complex scaffolds and development of advanced materials, RP provides a new avenue for cartilage repair. Computer-aided design and three-dimensional (3D) printing allow the fabrication of modeled cartilage scaffolds for repair and regeneration of damaged cartilage tissues. Specifically, the various processes of 3D printing will be discussed in details, both cellular and acellular techniques, covering the different materials, geometries, and operational printing conditions for the development of tissue-engineered articular cartilage. Finally, we conclude with some insights on future applications and challenges related to this technology, especially using 3D printing techniques to recapitulate the complexity of native structure for advanced cartilage regeneration.

Human mesenchymal stem/stromal cells (hMSCs) have been investigated and proven to be a well-tolerated, safe therapy for a variety of indications, as shown by over 900 registered hMSC-based clinical trials. To meet the commercial demand for clinical manufacturing of hMSCs, production requires a scale that can achieve a lot size of ~100B cells, which requires innovative manufacturing technologies such as 3D bioreactors. A robust suspension bioreactor process that can be scaled-up to the relevant scale is therefore crucial. In this study, we developed a fed-batch, microcarrier-based bioreactor process, which enhances media productivity and drives a cost-effective and less labor-intensive hMSC expansion process. We determined parameter settings for various stages of the culture: inoculation, bioreactor culture, and harvest. Addition of a bioreactor feed, using a fed-batch approach, was necessary to replenish the mitogenic factors that were depleted from the media within the first 3 days of culture. Our study resulted in an optimized hMSC culture protocol that consistently achieved hMSC densities between 2 × 105–6 × 105 cells/mL within 5 days with no media exchange, maintaining the final cell population doubling level (PDL) at 16–20. Using multiple hMSC donors, we showed that this process was robust and yielded hMSCs that maintained expansion, phenotypic characteristic, and functional properties. The developed process in a vertical-wheel suspension bioreactor can be scaled to the levels needed to meet commercial demand of hMSCs.