The challenges and considerations for emerging or future entrepreneurial researchers in microphysiological systems [version 1; peer review: awaiting peer review]

Microphysiological systems describe the use of divergent technologies to recapitulate complex physiology in vitro convergently in a cruelty and animal free manner. The technologies aim towards enabling researchers from academia and industry to conduct more ethical and cost-effective research and development, preclinical and translational, and to advance related fields such as precision medicine. However, projected markets appear relatively small compared to related markets, where regulatory implementation and reluctant end-user adoption creates uncertainty for the emerging technologies with associated technological maturity. Regardless of this, companies surpassed and expanded successfully beyond the predicted five-year survival rate through strategic technologyand business development through collaboration and partnerships. A hallmark of the companies is a core competency or unique intellectual property coupled with securing early investment and interest from industry role-players, using divergent strategies to create a burden-of-proof to encourage early adopter participation for technologies showing fit-for-purpose application. In this paper we aim to provide insights for the researcher who wants to become involved in the microphysiological field as an entrepreneur, requiring a generalized information landscape with keywords and concepts to expand their knowledge base. An overview is provided for the technological considerations for laboratory-tomarket product development, the current state of regulatory affairs and projected markets to provide a framework of reference to evaluate the randomly selected case study companies. Public information is used to provide company information regarding historical origin, funding, and technological strategies which secured funding as well as encouraged early adopter technology interests. Additional activities by the companies showcase that there is no single formulation for commercial survival five-years post-incorporation but a pattern, dictated by technology origin, to follow which for convergent or divergent opportunities in technology development Open Peer Review Reviewer Status AWAITING PEER REVIEW Any reports and responses or comments on the article can be found at the end of the article. Open Research Europe Page 1 of 26 Open Research Europe 2021, 1:38 Last updated: 01 JUL 2021


Introduction
Microphysiological systems (MPS) describe converging technologies aimed at recapitulating sufficiently complex physiology in vitro with increasing design diversity and application, where industry role-players lobbying for end-user product adoption and regulatory acceptance. Disruptive toolsets consisting of instrumentation, consumables and biological models' impact MPS technologies (i.e. organ-on-chip; organoids; microtissues; perfusion technology; microfluidics; etc.) to provide animal free, yet biologically relevant environments compared to the golden standards of the drug delivery pipeline (DDP) in a robust, reliable, more cost-effective, and ethical manner [1][2][3][4] . Global financial investments from the government and private sector drive the development of these technologies, where influence from the private and public sectors foster regulatory drafting and enduser technology adoption. Socio-political pressure requires the academia, pharmaceutical and biotechnology industry (APBI) to adopt more humane and ethical research and development (R&D) approaches using cost-effective and reliable methodologies, further serving as a driving force for MPS technologies as a replacement or companion technology for the golden standards of the DDP 1,5-9 . However, the burden-of-proof (BOP) required to demonstrate that MPS present with a fit-for-purpose (FFP) design requires a transparent and peer-reviewed approach to allow for the standardization consensus between regulatory bodies 5 where APBI contributes constructively. Unfortunately, a universal standardization to formulate regulatory guidelines is lacking for the technologies 2,3,10 . Additionally, a lack of broad-based adoption of MPS-based technologies in APBI can be attributed to the lack of standardization and technical limitations of the technologies when compared to the golden standards of the DDP, typically seen as emerging and young technologies with a small market when compared to loosely related technologies such as microfluidics (MF) 11-17 . It is not fully clear why the MPS market remains comparatively small, where factors such as regulatory acceptance and technology confusion may be contributing factors when coupled with slow technology adoption.
Regardless of the above challenges there has been a multitude of companies -start-ups and established -actively involved in bringing MPS technologies to market with active involvement in reducing the use of animal models, innovating with new technology offerings, or providing scientific services. Most of the companies were built around intellectual property (IP) or know-how generated at prestigious research institutions, where early commercial potential was identified and developed into a product or service by active engagement with early technology adopters and investors. This trend is continuing exponentially in the EU and United Kingdom (UK) where European Commission's (EC) Horizon-2020 initiative generously funds innovation towards developing state-of-the-art MPS technologies. Additional funding from the individual EU member states further encourages innovation to facilitate collaborative as well as innovative R&D in academia and industry to advance MPS technologies 1-4 by developing advanced in vitro biological models; physiologically relevant biochemical and biomechanical environments; machine learning-based image analysis and data management; as well as instrumentation and methodologies for automatization and workflow integration. Established and emerging entrepreneurs who wish to enter this market face technological, regulatory and market risks to demonstrate that their MPS technologies have a FFP design with a sufficient BOP in a comparable manner to the golden standards 18,19 , a daunting challenge when considered that the average start-up company lifespan is predicted to be less than five years for a small and highly competitive emerging market. However, global recognition that the DDP is neither sufficient nor efficient 20,21 for discovery or development of new therapeutics and very costly [22][23][24][25][26][27] has created an APBI end-user environment open to alternative solutions to facilitate cost-reduction 28-31 . Additionally, sociopolitical pressure is requiring the adoption of more humane and ethical research practises to forego animal research, where 3R (replacement, reduction, refinement) principle compliant methodologies aim to reduce overall cost and increase accountability in R&D. The conflation of circumstances has created the ideal opportunities for MPS to be the best solution.
In this article we aim to provide a primer for the new or emerging MPS entrepreneur in a broad-based manner using case studies framed within the context of technological considerations and regulatory challenges. This document is not intended as a definitive guideline; it is written from an entrepreneur and startup perspective active in this industry with the aim to provide knowledge to assist in further reading for concepts which may be unknown to the target audience. The entrepreneurial route is subjective, with success and failure regardless of textbook or instinctual strategies employed. Case studies are presented for randomly selected MPS companies, established and start-up, with a focus to IP history to implementation and expansion into a company to navigate the first five-year survival. Furthermore, the case studies are contextually framed by prefacing the reader with current information on: (i) the technological challenges required to create a FFP technology with sufficient BOP; (ii) the current state of regulatory affairs; and (iii) the current-to-projected MPS associated market sizes.

Methods
Typical scholarly searches were done to find information on state-of-the-art scientific information and for regulatory aspects using peer-reviewed literature, with other sources of information required to form an overview of the companies discussed in the text. The non-peer-reviewed data is described below. All data gathered was publicly available. No information was gathered for proprietary or non-public information, e.g. revenues or business strategies. Generally, the entrepreneur will identify market role-players as a means of assessing a market landscape report, followed by a technological (and other pertinent information) landscape report. An overview of the process follows. Non-peer reviewed information (i.e. press releases, blogs, protocols) provided by company websites typically do not have digital object identifiers (DOI) and rely on good faith.
Generating a market role-player report The companies showcased in this paper were selected from a market role-player landscape report (Table S3, Extended data 32 ) where data was gathered for the following parameters: company incorporation and history, product lines and services as well as intended customers or clientele. Companies were identified using general (www.google.com) and specific (https://www. crunchbase.com/; https://www.ventureradar.com/) search engines and databases using keywords, e.g. "organ-on-chip"; "advanced in vitro models"; "3D biological models"; "3D microtissues"; "microfluidics"; "microphysiological systems" individually or in combination.
Selection of case study companies for inclusion of additional parameters and creating a technology landscape report Companies were selected based on corresponding to our fields of expertise and commercial activities of instrumentation, consumables and biological models, thus excluding bioprinting and biomaterials companies. Companies where further selected having already passed a five-year period post-incorporation, excluding companies founded post-2014/15. Multinational companies (e.g. Philips, 3M) and distributors (e.g. Sigma Aldrich, Merck Millipore) who do not have a core business related to MPS were not considered. Final company selection was based on a randomized raffle of eligible companies remaining, followed by predetermined parameter directed data mining to expand on the datasets and condensed to Table 3 and Table S4 (Extended data 32 ). The predetermined parameters incorporated company history, founding technology patents or publications, historical and in-development product lines, collaborations, public declared funding and social media activity. Company specific resources references are pertinently mentioned contextually with references. Collected data was summarized in Table 3 and Table S4 (Extended data 32 ), provided with pertinent links and references providing the last date of access. The data collection and sources for the above is categorized as follows:

Data analysis
Agglomerated data was analysed using the OTTR (Observe-Think-Test-Revise) principles, filtered manually and crossreferenced to create Table 3, Tables S3 and Table S4 (Extended data 32 ). The companies' information was comparatively assessed followed by scientific and entrepreneurial interpretation. The data presented is not intended as an exhaustive data set, but as a guideline.

Technological challenges for designing in vitro MPS models: biological aspects
Challenges for MPS adoption VS golden standards of the DDP The Director of the United States (US) Food and Drug Administration (FDA) Centre for Drug Evaluation and Research, Dr Janet Woodcock, admitted that the DDP is broken especially in the context of clinical trials 20,21 . Lead development or discovery of drug candidates requires a process of evaluation; 2D in vitro > animal in vivo > human in vivo; with a low success rate (<15%) and astronomical costs (USD2.8 billion) over 13-15 years 5,6,22,23 . Clinical trials consume ≈65% of DDP costs, a crucial point for cost reduction and a driving factor in finding more physiologically relevant R&D models as well as predictive precision medicine approaches 23-25 . However, any improved in vitro models and methodologies with sufficient BOP and FFP design can provide companion or replacement technologies at any phase of the current DDP golden standards. A recent statistical evaluation 9 of MPS technologies concluded that up to 25% of R&D costs can be reduced as well as the turnover time in preclinical phases.
Advances in biomedical research -including the DDP -was shaped by the HeLa cell line 33 , but also embodies the challenges and limitations of 2D in vitro models 19,33,34 : (i) loss of phenotype and dedifferentiation; (ii) cellular depolarization; (iii) reduced gene and protein expression; (iv) altered membrane function; (v) altered metabolism; and (vi) cellular cross contamination. Animal models present with a non-human relevant physiology and genotypes 5,6,18,23 . Despite the intrinsic flaws, the DDP remains unchanged in a case of "the devil you know VS the devil you don't" where decades of investment in infrastructure, human capital development and academic consensus established regulatory processes reliant on the stability and functionality of the system for downstream cost-recovery of bringing new therapeutics to the market 26,27 . Cost recovery from the DDP process as well as socio-political pressures (e.g. 3R) has resulted in active downsizing pharmaceutical DDP activities with increased outsourcing to contract research organizations (CROs) -also a prudent financial and R&D risk mitigation strategy 28-31 . This ongoing process is changing the future of the cosmetics, agrochemical, food, and consumer goods markets in a similar manner, creating opportunities for new technologies such as MPS with added value additions 4-6,35 , especially when it comes to cost and turnover reduction 9 . The current challenge faced by MPS technologies is a lack of standardization with limited high throughput (HT) high content (HC) supporting technologies proving prohibitive in adoption for many APBI role-players [1][2][3][4][5][6]9 .
The initial lack of MPS technologies 9 fit within the "Diffusion of Innovation MPS biological model design -design for the end-user MPS biological models allowing for multi-cell type co-culture 42-44 , single-organ 45-48 or multi-organ approaches 49-52 and 3D printed tissues 53-57 , with or without tissue scaffolding 58-61 and micro-vascularization 62-64 present. Biological diversity in these models aim to recapitulate specific physiology but does not always clearly demonstrate the advantages or which golden standards is the target of comparison, a discrepancy between FFP design and supporting BOP 3-5,9 . The MPS in vitro model should clearly demonstrate sufficient complexity with complementary proof if the model is to act as replacement or companion model to the golden standards (Table 1). Figure 1 demonstrates the complexity for consideration when designing an MPS model. The proof required should provide demonstration of functionality with supporting peer-reviewed literature to encourage end-user adoption. From experience, the complexity for in vitro models differs across APBI, thus a "one size fits all" is counterproductive to foster end-user adoption. Engaging in a dialogue with the intended end-user can shape the ideal physiological complexity required for the intended application, allowing for strategic design choices. Typical questions which can contribute to improved end-user engagement can be compiled from Table 1 and Figure 1: (i) Will the model be used for disease modelling and development studies or as a HT-screening for 1 st round preclinical lead discovery from a compound library of thousands of molecules?
(ii) Does the model require (bio)chemical gradients or biomechanical stimuli?
(iii) Does the model require static or dynamic culture conditions with oxygen management? A minimum/ maximum culture duration?
(iv) The cost and time to develop the model (and prove advantages over DDP standards)?
(v) What will be acceptable costs for upkeep of the model (e.g. media formulations; hydrogels; consumables; etc.)?
(vi) What is the current available instrumental and laboratory infrastructure?
(vii) Will this be a single use model for novelty or for routine use? Could it be an easier route to provide a service delivery function to the end-user once the model is established?
The academic end-user may be grant dependent with a focus on publication frequency and impact seeking novelty, providing degrees of freedom for creativity in model complexity with inclusion of various companion technologies. Biotechnology and CRO companies present with research or service delivery specialization, where model design and allowances for companion technologies are dictated by the contract giver (e.g. pharma or other biotechnology companies) or regulatory requirements to advance research for commercially viable products. Pharmaceutical (and some biotechnology) companies may require HT and HC screening solutions where biological complexity should provide equal or better results than current in vitro 2D models but allow for facile integration into existing infrastructure and automated workflows. Examples of this will be discussed with reference to Figure 2, also considering Figure 1 as well as Table 1.

Example 1:
A pharmaceutical technologist needs to identify 10 most active lead candidate compounds from a 10,000-compound library for minimal liver toxicity. A two-dimensional monoculture (2DMC) in vitro model has the minimum required physiological complexity based on DDP recommendations with regulatory approval, where the methodology for HT-HC screening is already optimized to be cost-and time-efficient in an automated workflow. A MPS solution must demonstrate obvious advantages compared to the biological model, also demonstrating compatibility with the available and optimized infrastructure. The ideal model will be based on spheroids and organoids (SO) technology with added physiological complexity in a 3D environment, such as using extracellular matrix (ECM)-mimics and hydrogels, remaining compatible with HT-HC methodology 65-68 . The model can be complexed for later stage compound screens, where patient-derived cells from a biobank or primary isolation can be used in 2DMC or SO setups, progressing the molecules of interest through the preclinical screening process with more precision to the next stage of R&D.

Example 2:
A liver biopsy is taken from a patient to diagnose a liver pathology ( Figure 2). Apart from the histology-based diagnosis, some tissue can be used for various applications for

Relative cost (Low / Medium / High)
L M / H L / M H M / H † 2DMC -classical 2D monoculture; 3DCC -3D cell culture and co-culture; OOC -organ-on-chip; SO -spheroids and/or organoids; OBPCS -organotypicbiopsy-precision cut slices; ADMET -absorption, distribution, metabolism, excretion, and toxicology. the researcher from pure academic, preclinical or translation applications. The biopsy tissue can xenografted 80,81 or used to make patient specific-SO 66,73 , with the former allowing for the generation of SO as well. Alternately, the biopsy can be used as a precision cut slice or cubic biopsy samples 79,83 or preserved in a biobank 76,87 . The patient derived cells can be isolated into subtypes for SO generation or mixed into a scaffold for cellular self-assembly. The inclusion of ECM-mimics and hydrogels, dynamic or static culture conditions and desired (bio)chemical stimulus will be determined by the needs of the end-user. This type of approach where creativity is encouraged is typical for research institutes and well-funded researchers, where academic freedom allows for creating diverse solutions to advance state-of-the-art for models intended to be used for toxicology, disease modelling and precision medicine.

Accessorizing biological models: instrumentation and consumables
In vitro models require companion technologies to implement successfully, where interaction with the end-user will develop insight with regards to available infrastructure (e.g. CO 2 incubators, automated liquid handling systems, microscopy, etc.), the human capital and financial investment available as well as their ability and willingness to invest and expand. Securing interaction with the end-user will provide reciprocal insights. For the end-user, this provides awareness of technological offerings that can address the susceptibility of biological models with regards to temperature 88 and gas gradients 89-91 intrinsic to commercial humidified CO 2 incubators 88-91 . For example, we used such an approach to design a rapid heating/cooling microscopy stage solution to allow for the live imaging of various existing The complexity of the biological model will be determined by the end-user needs as well as the most common available infrastructure to the intended end-user. Academia is the most receptive to a variety of designs, which affords opportunities for novel discoveries and opens new research avenues. The pharmaceutical and biotechnology industries have less degrees of freedom, aiming for function over novelty, requiring design considerations for companion technologies or adherence to minimum requirement regulatory parameters. The complexity of a biological model will be dictated by biomechanical (e.g. shear stress, cell-cell and cell-extracellular matrix (ECM) interactions), biochemical (nutrient, metabolic waste and oxygen gradients), and physiological considerations (monoculture, coculture or 3D culturing techniques) to demonstrate an obvious advantage over the golden standards. ADMET -absorption, distribution, metabolism, excretion and toxicology. Compiled from 3-5,42-96.

Figure 2. A liver biopsy sample has many applications beyond diagnostics and pathology.
The biopsy tissue can be prepared as precision cut slices (PCS) used in direct ex vivo histoculture, rodent xenografted or used for isolating specific cell types which can be used in multiple tissue engineering approaches. Applications of these approaches can range from preclinical research to translational precision medicine.
C. elegans in various developmental stages as a function of temperature differentials using a perfused microfluidics chip. The identified niche was a lack of a standardized microscopy stage with universal compatibility. This was followed by developed companion technologies to expand on this initial niche, such as electrophysiological consumables for heart-on-chip applications.
Consumables and materials in direct contact or indirect (e.g. tubing material) contact with the biological model must be carefully chosen for characteristics such as biocompatibility, hydrophobicity, chemical resistances, leeching and gas permeability 97-99 . Polydimethylsiloxane (PDMS) is a cost-effective, popular, and versatile polymer used for MPS R&D despite being unsuited for biomarker and drug response-based research 3,35,100,101 , presenting with a challenge for industrialization. The high oxygen diffusion rate of PDMS can be found using other plastics with the inclusion of synthetic or biological membranes. However, most of the plastics do not allow for facile sterilization techniques or reuse, increasing the costs to the end-user. Consumable design should consider the advantages and limitations of materials inclusion such as: (i) design and efficient prototyping; (ii) manufacturing limitations and costs; (iii) the functional and aesthetic suitability for the end-user; (iv) sustainability and 3R compliance; and (v) cost to the end-user. Examples of these considerations can be seen in consumable design for the PhaseGuide TM designed OganoPlate™ from Mimetas, where this technology allows for liquid shear stress generation in vitro using static culture methodologies, but no biochemical gradients can be generated. Dynamic cell culture may require liquid or gas perfusion instrumentation (ElveSys, CN-Bio, CellASICS, etc.) to allow for cell culture gradient generation by mimicking blood flow and shear stress 2-4,102-107 , and culture durations of weeks or months. Commercial 102,103 and academic 104 advancements are demonstrating applications beyond individual MF chips or 24 multiwell towards HT-HC.
Regardless of the advancements and availability of standardised commercial consumables and instrumentation, well-funded researchers may opt to develop unique MPS-technologies. However, these technologies are geographically locked and not accessible to other researchers to independently evaluate or develop new technologies on these platforms 92-96 . The technological offering from MPS companies is not limited to biological models, consumables, and instrumentation where specialized biochemicals (e.g. hydrogels, culturing media), new biochemical and cell assays and data capture/analysis methodologies are needed for more comprehensive and faster turnaround analysis of organ-on-chip (OoC) and 3-dimensional cell culture and co-culture (3DCC) models. Current cell-based assays are adapted from 2DMC to 3DCC and OoC with less-than-ideal turnaround times for sample preparation and analysis, especially crucial when working with high priority samples or scarce samples.

MPS roadmaps for standardization and regulations
International socio-political pressure and expectation for APBI to adopt broad-based reductionist, ethical and cruelty-free R&D methodologies with improved transparency and communication also aim to improve the democratising of technology 8,108,109 . European initiatives such as the 3R 110 and the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines 6,111 have been adopted by the European Medicines Agency (EMA) 5 . The FDA -Defence Advanced Research Projects Agency (DARPA) -National Institutes of Health (NIH) MPS Program 112,113 and the MPS consortium signed under the Cooperative Research and Development Agreement (CRADA) has adopted similar initiatives. The USA Environmental Protection Agency has recently 8 declared that animal trials will be abolished by 2035, with similar initiatives following suit in the EU 114 . Europe has initiatives such as Human Organ and Disease Model Technologies (hDMT) 115 , Organ-on-Chip in Development (ORCHID) 116 and European Organ-On-Chip (EUROoC) 117 , which consist of well-respected industry and academic roleplayers to act as advisors for forging the regulatory guidelines for MPS in the EU. A challenge remains for the EU as the organizations tend to have geographic specific incentives and are not unified such as CRADA for the USA. The above initiatives can only reach their full effectiveness with full government, regulator and APBI interaction in a fully transparent process to support the drafting of comprehensive and sensical legislation. Thus far the efforts from these organizations have been building roads to animal-free research and adoption of MPS-based technologies. Companies have taken the responsibility to interact with regulatory agencies, where Emulate and CN Bio are actively in dialogue with the FDA (see MPS company comparative case studies: history and origin to current status) and in early 2021 announced FDA recognition 118 of their PhysioMimix™ technology with a joint publication of the use of the technology for drug safety evaluation. Similarly, TissUse 4 contributes by their CEO being actively involved in generating awareness with APBI as well as regulators. We have found that a dialogue with the FDA ensures that the most pertinent information for MPS technologies are available and influence interactions with role-players across APBI.
The 2017 EMA workshop 5 on "Non-animal approaches in support of medicinal product development -challenges and opportunities for use of micro-physiological systems" published the initial findings in 2018. The workshop was attended by prominent representatives from government and APBI to define scientific consensus, known facts and challenges of MPS technologies. Similarly, the EC's Joint Research Centre (JRC) 4,5,119 polled researchers across 26 EU/Eurozone territories, which echoed similar concerns as during the EMA workshop, where there was general cautious optimism. The conclusions are summarized for each sector in Table 2. The feedback received by the EC-JRC and EMA workshop reiterates that financial and technological risks contribute to an unwillingness in technology adoption, without complete technology aversion. The academics were the most critical of the technology when compared to the more muted responses from the pharmaceutical industry. The response from academia can be attributed to various factors but when considering that MPS technologies are diverse there can be confusion on choosing the technology proposition that will cater to their requirements.

Pharmaceutical industry Academia Government
• MPS has the potential to replace current preclinical models, in vitro and in vivo, preclinical models for ADMET and bridge knowledge gaps.
• MPS predict physiological responses better if designed well enough, being advanced in vitro models.
• MPS models must be designed FFP, with a defined BOP to clearly demonstrate the intrinsic limitations.
• MPS identified pharmaceutical targets with associated biological prediction has been promising with possible value addition for specific niches; and • MPS technologies are emerging but require maturation which can be assisted by developing new complementary technologies or using existing complementary technologies.
• MPS models are not always refined sufficiently for the target organ of interest, with uncertain genotype-phenotype relationship • Barrier systems and materials require improved biocompatibility and biomechanical relevance for the physiology to be recapitulated.
• The desire exists to combine individual OoC in series or parallel to better mimic in vivo conditions.
• Incorporation of immunecomponents and other blood components is essential, as well as more complex cocultures.
• The design of patient specific MPS, by using patient derived primary and stem cells.
• ADMET features (e.g. simulation of administration or sample taking) should be incorporated in MPS design.
• The function of the MPS models were not defined and too generalized • Application protocols deviate too much from standard laboratory and DDP protocols • MPS systems are relatively expensive for implementation and upkeep when compared to equivalent golden standards • 75% of respondents would adopt technology if these factors would be addressed • Undertakes to establish rules and guidelines with 3R approaches and inputs from APBI.
• Acknowledgement that technology advanced past the applicability of existing guidelines and regulations. Thus, new drafting is required.
• Clinical data should be free to access to determine relevance, where regulators should be consulted for confirmation thereof.
• The open sharing of all data is essential and strongly encouraged.
Regulatory approval, leading to drafting and adoption of guidelines for standardization of MPS technologies, appears to be a case of WHEN, not a case of IF. The slow adoption of innovative technologies is not always the norm, some technologies are adopted and implemented relatively fast.
MPS technology is currently in the converse of CRISPR technology, where CRISPR 120-122 addressed an (i) immediate and future scientific need in APBI in an innovative manner; (ii) by providing a novel and robust technology that resulted from a track record of core competency in the field of genetics and molecular biology; (iii) providing a technology that was readily comprehensible with a strong BOP and crystal clear FFP application; (iv) in a standardized and reproducible manner with acceptable financial investment for the end-user; (v) where the regulatory guidelines were already established by global scientific and ethical consensus. CRISPR is based on a robust track record of underlying well-characterized technologies with global use and implementation. The information provided thus far creates a perspective that MPS technologies require a standardized approach with ethical and legislative guidance to reach optimal maturation time. MPS technologies are intrinsically more complex collections of separate tools, each tool adhering to different standards, where the sum of the standards needs to adhere to equal strict ethical and scientific criteria. The inherent diversity in MPS technologies as well as human genetic diversity will be challenges for technology maturation and standardization.
The state of the market: are all opportunities obvious?
Market analysis for MPS is not trivial due to the related technologies (e.g. 3DCC; OoC; SO; tissue printing; etc.) and Table 3. Summarized comparison between MPS-related companies considering the initial core competency with the proofof-concept biological model, where the expansion strategy is provided. The perceived competitive advantages between the companies are given with the most probable technical or business challenges. All these companies have prestigious research institutions directly or indirectly involved in their history (Table S4, Extended data) and are in engaged in active collaborations across APBI with, thus these factors will not be used comparatively.

MPS company comparative case studies: history and origin to current status
Entrepreneurs in technology-based companies face a predicted five-year survival rate 157,158 . A recent publication focused on determining the factors of MPS and related technologies companies' longevity, determined that business models which presented large product and services catalogues coupled with perpetual new offerings risked failure attributed to overexpansion 158 . Furthermore, the authors identified company longevity parameters with the conclusion that hybrid (service and product) business models coupled with IP ownership fared better over the initial five-year survival period, with diverse target endusers in the food, pharmaceutical, cosmetics and (pre)clinical markets. Unique IP ownership is attractive to investors, especially in a start-up company where risk mitigation is key. Additionally, another contributing factor to company longevity was proven historical competency, usually a peer-reviewed academic track record, for the core technology and application. A contextual case study to be considered is Organovo 159,160 , founded in 2007, a liver micro-tissue bioprinting company that was listed on the New York Stock Exchange in February 2012 and valued at USD46.6 million 161 . Organovo attracted highly desirable collaboration interests from the pharmaceutical -and cosmetics industry such as MERCK 162 and L'Oreal 163 , attributed to company involvement in media coverages on 3R topics and reshaping the DDP 164 . The company expanded rapidly with project diversification to other microtissues dissimilar compared to initial core competency and faced multifaceted technical challenges and delivery delays, resulting in replacement of the management mid-2019 160 , where an intent to merge with Tarveda Pharmaceuticals Inc. was announced in February 2020 165a,165b . The technical challenges in biological model design ( Figure 1; Table 1) to deliver a sufficient BOP to advance the bioprinted tissues to viable products may have played a role, where the microarchitecture of organs differ significantly when factors such as biochemical gradients; biomechanical stimulus; cell population demographics; and degree of vascularization are considered. The appearance of market competitors such as CELLINK, Poeitis and Regemat3D displaced the lone wolf status of Organovo by providing similar or advanced technologies, providing the market with cost-effective technologies leading to competing products and services.
In the next few paragraphs case studies will be presented for selected MPS companies from a role-player landscape report (Table S3, Extended data 32 ), with a summary provided in Table S4 (Extended data 32 ) and Table 3. Publications and patents relating to the companies but not mentioned in the main text can also be found in Table S4 (Extended data 32 ). Data collection, usage and analysis is described in Methods.  (Table S4, Extended data 32 ) and webinar links, with a dedicated YouTube™ channel providing public access tutorials and application demonstrations. A joint webinar "3D OoC applications using AIM biotech chips" was held with Millipore Sigma, the latter provides the catalogue on their online store 172 . To date, the company provides 21 technology associated peer-reviewed publications (Table S4, Extended data 32 ) where various biological applications of the MF chip design is demonstrated, especially where microvascularization models are required for fluorescent microscopy-based projects.

AIM Biotech
Social media activity appears to be minimal with highest activity found on Facebook, mostly students directly interacting with Professor Kamm.

Ibidi GmbH
This privately held company was co-founded in 2001 by two PhD candidates from Technical University Munchen 173 , growing to an international industry role-player boasting more than 40 000 customers in 40 countries and >18000 publications associated with their product catalogue 173 . The impressive publication record associated with their products has created strong consumer confidence across APBI, with customer testimonials that include the pharmaceutical industry for a catalogue that includes biochips, consumables, biochemicals and instruments designated for analytics, temperature management and perfusion (e.g. liquid, gas) technologies. Ibidi describes itself as a company which caters specifically to the live cell 174-178 diagnostics and analytics markets, which correspond to USD1 billion and USD9 billion, respectively. Consumer education and resources are provided on the company website with text and video tutorials as well as protocols with active social media engagement to announce academic and industry advances in MPS and MF. Ibidi has ISO9001 and ISO13485 certification 173 for good manufacturing standards 179 and biomedical devices 180 , respectively.  196 , demonstrating perfusion culture is more dynamic for maintaining engineered liver tissues phenotypes over seven days, retaining physiologically more relevant activities than static culture conditions. Expansion on the initial core competency of perfusion culture liver microtissues saw a BOP creation using strategic collaborations (e.g. MIT) and funding from DARPA and the USA Department of Defence 189,190,193 . The in vitro models expanded progressively to include various types of 3DCC with addition of liver pathologies [195][196][197][198][199][200][201][202][203][204] towards proof-of-concept multiorgan approaches [205][206][207] . CN Bio holds the distinction of being the first MPS company to demonstrate a relatively successful 10-organ-on-chip technology for recapitulation of human-on-a-chip 2 . In March 2020, CITIC Securities, a Chinabased investment bank, announced a USD9 million investment for CN Bio business development in EU markets 208,209 . CN Bio has been featured in various forms of prominent media such as The Huffington Post 210 , BBC World Service 211 , Al Jazeera 212 , and the Discovery Channel 213 .

Emulate Bio
This Harvard-Wyss Institute spinout was founded in 2013 214 and to date holds a total capitalization of USD95 million 215 , the most invested in MPS company worldwide. The core technology is a lung-on-chip 216,217 device originating from the laboratory of Professor Donald Ingber. The core design of the MF-chip used for the lung-on-chip has shown application versatility to other on-chip microtissues [218][219][220][221][222] including organoid models 73,223 with associated pathologies [224][225][226][227][228] intended for disease modelling, drug discovery and absorption, distribution, metabolism, excretion and toxicology (ADMET) 229,230 . The "tattoo-on-chip" 231 collaboration with INTENZE Products demonstrated ADMET using skin-on-chip as a possible market for MPS technologies outside typical APBI, with the USA-FDA in the process of validating a liver-on-chip model for improved food safety 232-234 . Emulate created the instrumentation known as the "Human Emulation System", which comprises modular culture units as an alternative to using CO 2 incubators for their MF-chips. The company has an impressive workforce (Table  S4, Extended data 32 ) and actively engages diverse markets such as consumer chemicals, agriculture, personalized medicine, biotechnology as well as biomedical markets simultaneously. Furthermore, Emulate secured funding and collaboration with Johnson & Johnson, Merck, and The Michael J Fox Foundation for a thrombosis-on-chip 235 , asthma-on-a-chip 236, 237 and Parkinson's-on-a-chip 238 , respectively. An additional collaboration during 2020 with SpaceX included the human innervated intestine Chip (hiIC) MPS on a mission to the international space station 239 to demonstrate "automated maintenance, including imaging, sampling, and storage on orbit and data downlink for molecular analysis on Earth". Social media activity and end-user interaction is high, being the most active MPS company on the various media outlets (Table S4, Extended data 32 ). The company website (Table S4, Extended data 32 ) is well designed with a good user interface and provides access to publications, blogs, and forums with interaction with MPS experts and academics who have used Emulate products. Emulate changed the upper management structure in early 2020, appointing a new chief executive officer and chairman 240 . Social media activity appears minimal, where pertinent information is shared through newsletters and press releases on the company website.

InSphero
Organoid-based technologies are adopted more readily by APBI than other MPS-based biological models due to the technology being standardized, comparatively low-cost and readily integrated into manual or automated workflows 266,267 , where demonstrated clinical relevance for precision medicine can be realized 87, [268][269][270] . A strong BOP with near immediate implementation and market across APBI inspired InSphero to be incorporated based on patents 271,272 which lead to 3D Insight™ and GravityPLUS™ technologies, initially demonstrating standardised and repeatable production of liver-spheroids in a cost-effective manner for HT-HC methodologies for ADMET applications [273][274][275][276] . The company, in collaboration with the USA-NIH and National  291 , and pancreatic islets 292 . The product catalogue includes custom and commercial biological models that include general user protocols, new assay development protocols, product-specific consumables and proprietary biochemicals. The recent expansion of the product range includes the Akura TM inter-well liquid flow technology 293 to provide a competing technology to the OrganoPlate™ from Mimetas.

Mimetas
The PhaseGuide TM technology invented by Paul Vulto [294][295][296] led to the 2013 incorporation of Mimetas, leading to the development of the OrganoPlate™ consumable which provides quasi-perfusion of liquids using a rocking platform. A 2013 grant from the UK-based "CRACK IT" challenge enabled the development of a preclinical kidney-on-chip OrganoPlate™ model 297 for ADMET applications, further bolstered by a 2014 UK National Centre for the 3Rs grant where the judging panel comprised pharmaceutical industry experts (Roche-Pfizer-GlaxoSmithKline) 298 . A landmark collaboration occurred when Mimetas and Galapagos NV announced their intent -Galapagos was the first biotechnology company to adopt MPS technology as part of their core workflow 299 . Collaboration expansion during 2017 with Roche 300 and Molecular Devices 301 resulted in the development of a gut-on-chip model for the former and a consumer-ready HT-HC confocal microscopy product for the latter. Capitalizing on the HT-HC theme with the OrganoTEER TM instrumentation enabled real-time epithelial barrier function measurement 302,303 , providing faster experimental turnover than widely used transwell models. Mimetas increased their social media and end-user activity during 2019, which included sponsoring scientific conferences, symposiums, and workshops 304 mostly focused on SO-based biological models with their consumables. Crown Biosciences announced an exclusivity license (17 October 2019) on the OrganoPlate™ organoid platform 305 . An innovative biological model of snake venom-gland derived organoids demonstrated technology applications in biomining and antivenom biotechnology with a cruelty free and 3R approach 306 . Peer-reviewed publications for Mimetas-based technologies include kidney [307][308][309][310][311][312] , liver 313 , brain [314][315][316][317] , vasculature [318][319][320] , and intestinal 303 based models with expansion into cancer models 321,322 .
The Netherlands-based company expanded its geographic footprint to Maryland (USA) during 2019, conveniently located in the BioHealth hub where more than 800 life science companies and institutions as well as the USA-FDA and NIH are based 323 . Joss Joore, CEO and co-founder, gave a March 2016 TEDx Talk 324 , with further media coverage in Nature Medicine 325 , Science.com's "Supplier Insider" 326 as well as i-Micronews 327 . Social media activity is relatively high when compared to other MPS companies.

Summary
Hybrid business models 158 seem to be favoured by all the companies combining products, services, and consultation with sufficient online resources for the end-user. Furthermore, the companies secured early interest for collaboration or investment by providing technologies and core competencies that addressed the needs of APBI role-players such as automated workflow integration using HT-HC methodologies or providing improved in vitro models for ADMET and disease modelling, allowing improved investigation using microscopy methodologies ( Figure 3A and 3B). Additionally, companies expanded to the largest (USA) and fastest growing markets (Asia-Pacific), albeit at different stages in the company lifetimes. The younger companies (Table 3) display aggressive human resource expansion, marketing, and social media activity (Table S4, Extended data 32 ) than the more established companies, relative to time passed post-incorporation, possibly to secure larger market share as fast as possible, emboldened by generous funding (Table S4, Extended data 32 ). A comparison in the management structure of the companies, available on the respective Subsequently, the business model will need to be refined on a continuous basis in response to competitors, funding, and other factors (B). The initial core competency (A) will form the foundation for the business and developing market appetite (B), where reciprocal feedback loops will help navigate the five-year survival rate (C). There is no single route to guarantee successful five-year survival post-incorporation, but adaptability is essential. IP -intellectual property.
websites, correlates overall company behaviour with cultural and generational composition in the management [328][329][330] : older generations tend to favour defined job portfolios with high riskaverse behaviour, valuing direct peer-to-peer networking to establish longer lasting professional relationships. Conversely, individuals from younger generations prefer more diverse job portfolios with higher risk-taking behaviour, using social media tools to disseminate information and establish more superficial professional relationships. The strategies used for product rollouts and technology expansion of the companies echo this strategy as well.  (Figure 3). Established companies can, however, create negative sentiment and cause near market collapse such as the gaming industry during the early 1980s, attributed to the gaming pioneer and then dominant market role-player Atari 334,335 . A movie tie-in video game loosely based on the 1982 Steven Spielberg movie "E.T. -the Extra Terrestrial" saw 3.5 million copies unsold, near three times more than sold, then buried in a landfill.
The true impact of social media and other online platforms on company business development and marketing is unclear. YouTube is mostly used for posting marketing materials, technical tutorials, and webinars (  (Table 2), technology readiness and end-user confidence, regardless of the international socio-political pressure to adopt animal-free 3R methodologies. Consumable-focused companies (AIM, Ibidi, Mimetas) face a reduced BOP to demonstrate product TRL by showcasing a primary obvious advantage ranging from ergonomic design, improved microscopy applications, HT-HC compatibility, or the ability to mimic physiological occurrences. The BOP is expanded upon by extensive publications from sold products or collaboration. Interestingly, consumable companies are reported as part of the bioanalytics and microscopy markets, larger markers than OoC/MPS, regardless of their prominent roles in MPS/OoC across APBI. Added value addition to consumable products can include companion instrumentation and biochemicals, but there is a high reliance on collaborative partners from APBI to provide peer-reviewed BOP. The technical challenges ( Figure 1; Table 1) to validate a novel biological model with an added companion technology (i.e. perfusion technology) comparatively to a representative golden standard is an exhaustive process, requiring a BOP for both components individually and in tandem to advance to an acceptable TRL to foster technology adoption. The gradual progression to demonstrate TRL can interest the early APBI adopter or investor at any stage of the technology development; however, this is a grand challenge when the targeted end-user sentiment is considered (Table 2). This is a challenging situation for the entrepreneur and company as investors typically require a return on investment in a relatively short duration (<5 years). Implementation of service delivery using the MPS/OoC technologies can serve a dual function of promotion and a source of revenue, especially if done in strategic collaboration. The joint publication from the US-FDA and CN Bio for an in vitro liver model 194 using the PhysioMimix™ system has given the latter a higher level of trust in their technology for applications in APBI and from current as well as future investors. Emulate 232-234 has used a similar US-FDA interaction for advancing in vitro liver models in a market that is highly coveted yet oversaturated with commercial and academic models. A PubMed search [Last accessed 15 December 2020] using keywords "in vitro liver" yields ≈20 000 publication for the past five years, ≈4400 publications for 2020. In vitro liver models face similar challenges as other in vitro models (cell contamination; genetic shift; non-physiological cell densities) but more specific challenges are providing liver sinusoidal heterogeneity with oxygen zonation, where correlation with drug oral bioavailability and metabolism is lacking 348 . A conservative estimate suggests ≈50 commercial in vitro liver models (internal database), where a recent review 349 highlighted the creative heterogeneity for in vitro liver models including the most prominent MPS/OoC companies in 2019 providing models with application-defined peer-reviewed publications. The companies included Emulate, CN Bio, InSphero and HµRel -FDA recognition provides a clear competitive advantage (Figure 3) for two companies from the list when considered from an end-user perspective.
The above companies used different strategies post-incorporation to mitigate risk to an acceptable level while attracting interest and adoption from APBI and early investment based on an initial core competency coupled with scientific merit. Active campaigning with APBI, regulatory bodies and the public through media outlets is creating an appreciation and awareness for the role of MPS in advancing 3R implementation in a cruelty free manner. Technology maturation is advancing within the uncertain market where technology reputation is crucial. A cautionary case-study for any bioscience entrepreneur should include Elizabeth Holmes, founder of Theranos 118,350,351 , where the meteoric rise of the company was based on perceived core-competency and novel IP in diagnostic MF. Through reputation and networking from the high-profile board of directors, vast investment was secured, based on future-technology without a robust peerreviewed BOP or current scientific merit. Elizabeth Holmes is currently facing charges of fraud and investors who want to recuperate their investment 352 .

Future outlooks
MPS-based companies enter an emerging market with uncertain projected future value, the true market value currently supressed by non-standardised technologies, regulatory process, and slow adoption by APBI end-users. However, progress made by leading MPS companies and regulatory bodies are progressively changing a roadmap to a charted journey, defining MPS requirements for standardization, in collaboration with APBI to shape the industry. Biological models and MPS technologies that have already received recognition from regulatory bodies are well on their way to become MPS reference standards. Consumables-based companies face less regulatory barriers but are active in a competitive market where technical advantages are not always obvious. Regardless, projected future MPS market size remains relatively low where technology maturity is adoption prohibitive for the greater academic community, especially in developing countries, where reliance on wellestablished methodologies or collaboration with more wellfunded research groups are cost-effective. A further challenge for MPS companies is the continuous R&D downsizing and outsourcing seen for the biotechnology and pharmaceutical industry, coupled with a reluctance to invest in emerging technologies regardless of potential. A select few companies successfully demonstrated HT-HC workflow integration, but challenges remain in proving physiological relevance for the intended applications. The convergence of the above challenges created new opportunities for MPS companies to provide CRO services, benefitting the MPS industry in the long-term: (i) MPS-technologies are provided in a reduced risk and more cost-effective manner to a larger user-base.
(ii) A larger user feedback can assist in improving MPStechnologies by identifying minimal universal requirements, particularly when animal-free methodologies are to be adapted.
(iii) A potentially more extensive BOP can be created to generate TRL confidence for MPS-technologies, for potential investors and end-users, leading to improved industry standardization.
(iv) Larger acceptance of MPS-technologies will eventually evolve into more affordable alternatives for democratising state-of-the-art research.
The companies in this article progressively advanced the MPS field commercially and technologically, paving the way for a newer generation of researchers and entrepreneurs. Regardless of the current challenges the future of the technologies is an untapped potential to technology standardization and global democratising of research using animal-free methodologies. The future of the per definition MPS-market (see section The state of the market: are all opportunities obvious?; Table S1, Extended data 32 ) is projected to be less lucrative than related markets of MF or cellular analytics, but MPS are per definition a multidisciplinary field (Figure 4) reliant on a complex confluence of companies with innovative technologies to recapitulate physiology in vitro. The future entrepreneur must view the MPS industry as a complex environment that appears oversaturated; however, a basic keyword search using Google (Figure 4) highlights by SEO what end-users will find when searching for a company with associated services. A recent market projection 353 of the top OoC/MPS companies that will directly influence the pharmaceutical industry highlight established and new companies entering the market with enthusiasm and outpacing some established competitors. There is a segment of the MPS market that will eventually require animal-free growth media to fulfil true compliance to ethical and cruelty free 3R approaches, where currently there is an overreliance on animal-derived serum. An additional consideration for avoiding animal-derived components, especially in foods and medicine, include the costly removal of viruses and prions while reducing overall antibiotic usage. Developing an effective media formulation will not only serve the MPS industry but also the synthetic meat 156 and cell therapy 354 industries, which are projected at USD30 billion and USD6.7 billion, respectively, by 2030. The composition of media includes many biomolecules such as growth factors and hormones that will have to be sourced from non-animal origin such as with insulin, resulting in a significant increase in the biomolecules market in the 1990s. Similar arguments can be made for developing new MPS bioassays to improve experimental turnover or artificial intelligence (AI)-based analysis platforms for real-time culturing adaptation, data capture or analysis. Considering that current in vitro models have still to provide sufficient complexity to replace animal models or to advance precision medicine, new technologies and methodologies can contribute significantlyespecially for plug-and-play ready cells in native-mimicked scaffolding with cryo-storage or biobanking design. Entrepreneurs can contribute in any of these fields to advance the maturation and commercial applications of MPS, where creativity and adaptability (Figure 4) will be the guiding factor to navigate the five-year post-incorporation survival.

Data availability
Underlying data All data underlying the results are available as part of the article and no additional source data are required. This project contains the following extended data in the file 'Extended Data.docx': •