Guest article by Viraj Mehta – Organ-on-chip classification system (OoCCS): Benefits and limitations

Over the past decade, the field of microphysiological systems (MPS) has experienced a significant growth, driven by extensive validation studies conducted by academia, industry, and organ-on-chip manufacturers. In particular, the last 4 years witnessed at least two-fold increase in industrial validation of organ-on-chips [1]. However, the majority of these industrial studies were exploratory in nature, and the MPS data generated were not included in regulatory filings. The recent IQ-MPS survey report also expressed the cautious approach of the industries in including the MPS data in the regulatory filings [2].

Lack of regulatory guidelines, low-throughput of the devices, and lack of standardization are some of the significant roadblocks against MPS adoption in industries. Several initiatives were taken by regulatory authorities, various government bodies, and organ-on-chip companies to address these limitations. A manuscript series published by IQ-MPS group of scientists provided detailed guidelines for the usage of MPS models for safety assessment of compounds in the drug discovery [3]. Several organ-on-chip developers have introduced 96- and 384-well formats of their devices to overcome the low throughput of the systems. Additionally, these conventional well-plate formats are being validated for compatibility with robotic fluid handling platforms, aiming to reduce human intervention and meet industry standards. Finally, there have been numerous efforts worldwide to standardize terminology, processes and systems involved with organ-on-chips [4]. However, with the emergence of various commercial organ-on-chip companies, a standard classification system is required [5].

Various categories of standard classification system of organ-on-chips

The primary advantage of organ-on-chip systems is their ability to incorporate various organ-relevant cells to replicate the multicellular spatial arrangement of an organ. Commercially available organ-on-chips can be broadly classified into four categories based on their unique designs and working principles that enable spatial patterning of cells: 1) membrane-based, 2) capillary pinning or micro-compartmentalization or micro-patterning based, 3) sacrificial or non-sacrificial materials based, and 4) Milli-compartments (or milli-wells) or micro-compartments (or micro-wells) based. In this classification, the high-throughput versions of organ-on-chips developed in conventional well-plate formats have been categorized according to the working principle of single functional unit. This is to prevent the misclassification of such high-throughput plate-versions into the last category of milli-compartments (or milli-wells) or micro-compartments (or micro-wells) based category. Figure 2 illustrates the classification of various commercial organ-on-chip platforms into specific defined categories.

Category 1: Membrane-based

• • Several devices in this category can be considered as a dynamic version of conventional transwell cultures, since they use microporous membrane-based compartmentalization, allowing the application of fluidic shear stress in at least one channel. Additionally, similar to transwells, these devices are compatible with culture of various barrier tissues.
  • Generally, the membrane plays a major role in facilitating the cell adhesion, and it is generally permanently bonded to the chip materials.
  • These devices often involve mechanical stimulation to mimic breathing motion, gut movement, and joint movement. The membrane transfers mechanical stimulation to the cultured cells in contact with it.

Category 2: Capillary pinning or micro-compartmentalization or micro-patterning-based

• • Capillary pinning is defined as confinement of fluids due to an abrupt change in capillary pressure
  • These systems are generally compatible with hydrogel encapsulated 3D cultures
  • Additionally, they are suitable for in vitro vascularization using co-culture with endothelial cells.
  • This method of micro-patterning or micro-compartmentalization is very popular among organ-on-chip developers focused on neuroscience related applications.

Category 3: Sacrificial or non-sacrificial materials based

• • Devices in this category allow incorporating tubular structures inside them using:
    • Non-sacrificial materials such as glass mandrel, steel needles, or 3D bioprinting based photocuring of non-sacrificial materials
    • Sacrificial materials such as pluronics, gelatin, PVA, and PEGDM. Additionally, 3D bioprinting can also be utilized to deposit sacrificial materials in a particular fashion.
  • The resultant tubular structures can be endothelialized or epithelialized and cultured under dynamic flow.
  • Hence, these devices are suitable for mimicking kidney tubules and gut lumen for various ADME and toxicology applications.

Category 4:  Milli-compartments (or milli-wells) or micro-compartments (or micro-wells) based

• • This category can be subdivided into open-compartment (or open-well) and closed-compartment (or closed-well) designs.
  • Several open-well configurations introduced by organ-on-chip companies are versatile in nature and compatible with variety of cell culture methods including those based on transwells, 3D scaffolds, membranes, 3D spheroids, 2D monolayer, reconstructed tissues, and 3D hydrogels.
  • Closed-well designs generally trap tissues inside the microwells or milliwells after complete assembly, making them less versatile compared to open-well designs. Additionally, open-well configurations generally also allow retrieval of tissues for performing immunohistochemistry, flow cytometry and other molecular studies
  • Open-well systems are generally suitable for multi-organ interaction studies and include on-chip integrated pumping system for recirculating the culture medium.
  • However, it should be noted that such devices may not always follow conventional well-plate formats and dimensions.

Benefits of standard classification system of organ-on-chips

There are four key benefits of standard classification system, as discussed below:

Limitations and future perspectives

With the current pace at which the field is moving, many new technologies will emerge that may blur the lines between categories, creating overlaps that are not easily classified. For instance, technologies from Chiron and Biomimix had to be classified under both the categories 1 and 2 since they involve membrane and operate based on principle of capillary pinning. Such hybrid systems that combine features from multiple categories will become increasingly common and cannot be confined to a single category. Another limitation is that the proposed classification primarily focuses on the physical and architectural aspects of the chips, potentially overlooking their biological and functional characteristics. Moreover, there can be other multiple ways to categorize organ-on-chips. Some of the possible ways include: (1) single-organ on chips and multi-organ-on-chips, (2) based on type of cells used, such as cell lines, iPSCs, and primary cells, (3) based on the organ cultured, (4) type of material used for the fabrication of chips, and (5) based on the context of use. Hence, careful consideration and discussion with relevant stakeholders will be required to finalize the categories of classification.

As discussed before the standard classification system will potentially help in developing category relevant additional guidelines and standards for a specific context of use. For instance, categories 2 and 3 are mainly based on matrix-embedded culture of cells. Hence, parameters such as matrix thickness, matrix composition, matrix mechanical properties, permeability, porosity, and drug binding factor should be standardized for a specific context of use. Similarly, additional guidelines can be developed for category 1 devices that often include mechanical stimulation in the form of stretch. Furthermore, physical parameters such as membrane thickness also need to be standardized for category 1 devices. Fluidic shear stress can also be standardized based on the categories defined for a specific CoU. However, flow rates applied to realize the standard shear stress will be different for the four categories, which can be a part of category specific standard guidelines. Finally, additional guidelines will be required for multi-organ-on-chips (especially for category 4) with recommended standard scaling approaches used to design them.

Category specific physical and operational guidelines can be developed based on the standard classification, but biological functional guidelines will most likely remain common for all the categories for the majority of CoU. IQ-MPS consortium developed three stage guidelines for safety assessment based on functional characterization of liver-chips including albumin production rates, urea production rates, and metabolic enzymes activity [6]. Hence, organ-on-chip devices from all four categories will have to meet these functional guidelines in order to get qualified for safety assessment CoU. In conclusion, the standard classification system will significantly help in developing regulatory guidelines, standards, educational and training materials, and in overall development of the field.

References

[1] Viraj Mehta, How Industry Embraces Organ-on-Chips: A 2024 Status Report, BioPharmaTrend (2024). https://www.biopharmatrend.com/post/785-organ-on-chips-advance-to-industry-a-comprehensive-report-2024/ (accessed June 4, 2024).

[2] T.K. Baker, T.R. Van Vleet, P.K. Mahalingaiah, T.S.P. Grandhi, R. Evers, J. Ekert, J.R. Gosset, S.A. Chacko, A.K. Kopec, The Current Status and Use of Microphysiological Systems by the Pharmaceutical Industry: The International Consortium for Innovation and Quality Microphysiological Systems Affiliate Survey and Commentary, Drug Metab Dispos 52 (2024) 198–209. https://doi.org/10.1124/DMD.123.001510.

[3] K. Fabre, B. Berridge, W.R. Proctor, S. Ralston, Y. Will, S.W. Baran, G. Yoder, T.R. Van Vleet, Introduction to a manuscript series on the characterization and use of microphysiological systems (MPS) in pharmaceutical safety and ADME applications, Lab Chip 20 (2020) 1049–1057. https://doi.org/10.1039/C9LC01168D.

[4] D.R. Reyes, M.B. Esch, L. Ewart, R. Nasiri, A. Herland, K. Sung, M. Piergiovanni, C. Lucchesi, J.T. Shoemaker, J. Vukasinovic, H. Nakae, J. Hickman, K. Pant, A. Taylor, N. Heinz, N. Ashammakhi, From animal testing to in vitro systems: advancing standardization in microphysiological systems, Lab Chip 24 (2024) 1076–1087. https://doi.org/10.1039/D3LC00994G.

[5] M. Piergiovanni, S.B. Leite, R. Corvi, M. Whelan, Standardisation needs for organ on chip devices, Lab Chip 21 (2021) 2857–2868. https://doi.org/10.1039/D1LC00241D.

[6] A.R. Baudy, M.A. Otieno, P. Hewitt, J. Gan, A. Roth, D. Keller, R. Sura, T.R. Van Vleet, W.R. Proctor, Liver microphysiological systems development guidelines for safety risk assessment in the pharmaceutical industry, Lab Chip 20 (2020) 215–225. https://doi.org/10.1039/C9LC00768G.

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