Challenges in Scaling Up 3D Cell Culture for Industrial Use: Navigating Hurdles

Introduction

Three-dimensional cell culture (3DCC) has emerged as a powerful tool in the field of biomedical research, offering a more physiologically relevant environment compared to traditional 2D cell cultures. 3DCC technologies, such as spheroids, organoids, organ-on-chip (OoC), microfluidic tissue systems or tissue chips, have opened new vistas in pharmaceutical research and industrial applications. As researchers strive to bridge the gap between in vitro and in vivo conditions, the demand for scalable 3DCC systems for industrial applications has grown exponentially. However, the transition from lab-scale exploration to large-scale industrial implementation brings forth a unique set of challenges that must be addressed to ensure the reproducibility, reliability, and efficiency of the process.

We'll delve into the intricacies of scaling up 3DCC and explore the hurdles, drawing insights from research articles and real-world examples.

Navigating Hurdles

Organoid Complexity and Scalability

The inherent complexity of organoids, designed to mimic the intricate structure and function of organs, poses a significant challenge in scaling up. Achieving reproducibility and scalability without compromising the physiological relevance of these 3D structures is a multifaceted task.

• The pioneering work by Hubrecht Organoid Technology (HUB) in establishing a biobank of organoid cultures showcases the potential for scalable, patient-derived organoid models. However, challenges persist in optimizing culture conditions for large-scale reproducibility.
• The work of Lancaster and Knoblich in generating cerebral organoids highlighted the potential for recapitulating brain development and human-brain-like microanatomy. However, scaling this complexity to an industrial setting necessitates advancements in culture techniques and bioreactor design.
• Research by Clevers and colleagues demonstrated successful scaling of organoid cultures for high-throughput drug screening, highlighting the importance of optimizing culture conditions for reproducible results on an industrial scale.

Spheroid Uniformity and Nutrient Distribution

Spheroids, simpler in structure compared to organoids, demand meticulous attention to culture uniformity when scaled up. Ensuring homogenous cell distribution, nutrient supply, and optimal conditions throughout the culture process are vital for reproducibility.

• The automation-driven approach adopted by companies like InSphero, specializing in 3D cell culture technologies, demonstrates advancements in high-throughput spheroid production. However, challenges remain in optimizing parameters for consistent large-scale production.
• The study by Edmondson et al. on spheroid cultures emphasized the importance of optimizing parameters like culture media and seeding density for consistent spheroid formation. Scaling up demand’s advancements in automation and optimization techniques.
• Studies exploring large-scale spheroid production in suspension cultures, like the work by Hirschhaeuser et al., underscore the importance of developing scalable systems to maintain uniformity and maximize nutrient delivery.

Organ-on-Chip Engineering, Microfluidics, and Integration

The transition of organ-on-chip (OoC) and microfluidic systems from lab to industry is challenged by intricate engineering requirements. Consistent flow, nutrient distribution, and maintaining consistent micro-physiological functions at a larger scale necessitate innovative engineering solutions. The transition of OoC systems from lab to industry is challenged by intricate engineering requirements.

• Emulate, a company focusing on several OoC technologies such as liver, kidney, lung, colon, duodenum, etc, has made strides in recreating the microenvironment of organs for drug testing. However, challenges persist in integrating these systems seamlessly into large-scale industrial processes.
• The lung-on-chip technology developed by Huh et al. showcased the potential to mimic organ-level functions. However, industrial-scale implementation requires further breakthroughs in system design, integration, and robustness.
• The Wyss Institute's lung-on-a-chip technology, developed by Ingber and colleagues, showcases the potential of microfluidic systems for mimicking organ-level functions. However, industrial-scale implementation requires further advancements in system design and integration.
• Dolomite Microfluidics, a global company specializing in microfluidic solutions, exemplifies advancements in precise fluid control. Yet, challenges persist in adapting these technologies for large-scale industrial applications.
• Research by Beebe and Young on microfluidic cell culture platforms provides insights into precise control over the microenvironment. To transition these technologies to an industrial scale, optimizations in design and automation are imperative.
• Research by Bhatia and Ingber on microscale technologies for controlling cell behavior provides insights into the potential of microfluidics. However, translating these concepts to industrial settings demands innovations in technology and process optimization.

A major challenge of integrating OoC systems is the low-throughput character of cell introduction. The initial preparation of OoC systems and the injection of cells is often still a rather manual process. Cell suspensions or cell-loaded hydrogels are pipetted into individual inlet ports and pumped to the desired culture site. Standardization and automation are critical, but complicated due to the importance of sterile cell handling, avoidance of stress, and short time windows. Additional challenges include the correct scaling of organ and tissue sizes as well as cell numbers that must be considered so that active cell ratios are physiologically relevant, and responses to stimulations are accurate.

Other difficulties include liquid handling, material compatibilities, monitoring systems, and parallel experimentation. Integrating microfluidic technologies into large-scale processes introduces challenges related to system design, precision, and scalability. Critical considerations include efficient management of fluid dynamics, avoiding shear stress, and ensuring continuous nutrient supply. Integrating microfluidic technologies introduces challenges related to system design, precision, and scalability.

Some key challenges for integrating different OoC platforms include biological challenges such as appropriate scaling of organ sizes and cell numbers, creation of a universal media for perfusion of different cell types, iPSC cell sourcing, vascularization of tissues, inclusion of immune components, as immune responses can shift drug dose responses and consideration of circadian and other cycles on cells. In addition, technical challenges include drug adsorption and its binding to the highly lipophilic surface of PDMS, the connection of platforms to maintain sterility and avoid bubbles, flow rate differences between platforms, the inclusion of biosensors, and creating ideal oxygenation and nutrient levels for different organs and automation of the whole system for high throughput screening while industrial validation.

The last but major challenge is getting a consistent non-animal source of biomaterials or bioinks on a large scale while eliminating batch-to-batch variation. Biomaterials such as Matrigel, geltrex, cultrex, rat tail collagen, etc. which are available in the market and are widely used, not only have an animal origin but also involve unethical practices of extraction, and might contain contaminants as well as batch-to-batch?variation.

'There are no big problems, there are just a lot of little problems' is one of the most inspirational quotes by Mr. Henry Ford. Here in this article, the little problems have been listed. With the pace at which advances are being made, there is no question that 3D Cell Culture will hold a huge space in the market shortly.

Article by Deepa Chaturvedi , PhD Scholar, Nanomedicine Research Group, ICTMumbai .