Bovine colon organoids: From 3D bioprinting to cryopreserved multi-well screening platforms

Elfi Töpfer, Anna Pasotti, Aikaterini Telopoulou, Paola Italiani, Diana Boraschi, Marie-Ann Ewart, Colin Wilde

Abstract:

Three-dimensional (3D) colon organoids, termed “colonoids”, derived from adult stem cells represent a powerful tool in in vitro pharmaceutical and toxicological research. Murine and human colonoid models exist. Here we describe the establishment of bovine colonoids for agribiotechnological applications, and extend the repertoire of colonoid culture options through proof-of-principle for bioprinting and novel in-plate cryopreservation technology. As a first step, we differentiated established long-term bovine colonoid cultures into mature colonoids. Tissuespecific differentiation was demonstrated by gene expression. Second, we investigated cryopreservation of colonoids in situ within an extracellular matrix in multi-well plates. Upon controlled thawing, cryopreserved 3D cultures grew at similar rates to unfrozen colonoids. Cytotoxic sensitivity to staurosporine was not significantly different between in situ freezethawed and unfrozen control cultures. Third, scalability of colonoid culture assembly by extrusion bioprinting into multi-well plates using GelMA bioink was assessed. With optimised bioprinting and crosslinking parameters, colonoids in GelMA were printed into 96 well culture plates and remained viable and proliferative post-print. For tissue-relevant in vitro studies we furthermore established differentiated colonoid-derived monolayer cultures on permeable membranes. Taken together, we outline novel in vitro approaches to study the ruminant colonic epithelium and introduce cryopreservation.

Keywords: bovine enteroid, monolayer culture, 3D bioprinting, in-plate cryopreservation organoids

Introduction

Intestinal epithelial cell culture methods have made a quantum leap in recent years, with the introduction of self-organising, adult stem cell-derived epithelial organoid cultures (1). Intestinal in vitro organoids, called enteroids, are three-dimensional (3D) multicellular structures that resemble their in vivo counterparts much more accurately than traditionally-used cell lines such as Caco-2 (2).
Enteroids have been established from multiple tissue sites, such as the oesophagus (3), stomach (4, 5), small and large intestine (1, 6, 7), and from various species such as chicken, mouse, human, and pig (1, 8, 9). In order to establish an expanding enteroid culture, single sorted Lgr5+ stem cells or Lgr5+ stem cellcontaining intestinal crypts are embedded in an extracellular matrix (ECM), and critical niche factors that activate Wnt and EGF signalling are provided in the culture medium (10). Under optimal culture conditions, enteroids expand limitlessly and are genetically stable (1, 11). Upon withdrawal of niche factors, enteroids undergo differentiation along all tissue-specific lineages, representing the cellular diversity of their tissue of origin (1, 7).
Enteroid and, in general, organoid technologies have been used in several different applications, ranging from high-throughput drug sensitivity and toxicology screening (12-14) to the establishment of functionally sophisticated monolayer models on permeable membranes (7, 15, 16). Furthermore, organoid applications in tissue engineering have been discussed (17), and the first defined designer matrices have been described as an alternative culture environment to the commonly-used Matrigel (18).
These developments have mostly focussed on murine and human tissue-derived organoids. In this study, we aimed at introducing this culture technology into livestock industry-related research, where great interest lies in ensuring animal health and productivity. Bovine enteroids are of particular interest, as cattle are a major asset of the livestock industry, with over 7 million tonnes of bovine meat and over 160 million tonnes of cow’s milk being produced annually in the European Union (EU-28) alone (19). Up until now, in vitro modelling of bovine epithelial barriers has relied largely on immortalised or cancer-derived cell lines, or on short-lived primary cultures prone to epithelial–mesenchymal transition (EMT) (20-23). Here we describe culture and differentiation conditions for bovine colonic organoids (colonoids). We outline how the established bovine colonoid culture can be used in various in vitro systems, ranging from extrusion bioprinting to monolayer development. In addition, we demonstrate that 3D colonoid cultures are suitable for adaptation to multi-well screening platforms in combination with in-plate cryopreservation. This enables uncoupling of production from analytical use, and convenient end-user introduction into medium-throughput screening activities, e.g., in toxicological in vitro studies.

Material and Methods

Crypt isolation and colonoid culture

Bovine colon tissue specimens were obtained from a local abattoir with a certified negligible risk for BSE. Crypts were isolated from washed, dissected epithelial strips according to published protocols (1). For seeding, 500 crypts/well were resuspended in Matrigel® – GFR, phenol red-free (Corning, Bedford, USA), and seeded as 35 µl centric domes into a 24-well plate. After a 20 min polymerization period at 37°C, 500 µl of expansion medium was added per culture well. Optimal bovine colonoid expansion medium contains advanced DMEM/F12 (GIBCO, Life Technologies, Paisley, UK), 1% BSA (Sigma-Aldrich Inc., St. Louis, MO, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich), 2 mM L-glutamine (SigmaAldrich), 1x N2 supplement (GIBCO), 1x B27 supplement (w/o vitamin A) (GIBCO), 10 mM HEPES (Sigma-Aldrich), 50% Wnt-3A conditioned medium (from L Wnt-3A cell line ATCC® CRL-2647™ according to provider’s instructions), 1 µg/ml hrR-spondin (PeproTech, Rocky Hill, NJ, USA), 100 ng/ml hrNoggin (PreproTech), 50 ng/ml hrEGF (PreproTech), 500 nM A-83-01 (Sigma-Aldrich), 10 µM SB202190 (Sigma-Aldrich), 10 nM [Leu]15-gastrin-1 (Sigma-Aldrich), 10 mM nicotinamide (Sigma-Aldrich), 1 mM N-acteylcysteine (LKT Laboratories, St. Paul, MN, USA); 5 µM CHIR99021 (Sigma-Aldrich); 10 µM Y-27623 dihydrochloride (Cayman Chemical, Ann Arbor, MI, USA); 50 nm PGE2 (SigmaAldrich); and 2 mM sodium acetate (Sigma-Aldrich). Colonoids arising from embedded crypts were passaged in a ratio of 1:3 every 4-5 days, adapting a published protocol (24). While in 24-well format colonoids were cultured in 35 µl Matrigel domes with 500 µl medium (see above), 96-well cultures contained 6 µl Matrigel domes and 100 µl medium. For differentiation trials, cultures were washed with PBS (w/o Ca++ and Mg++) (GIBCO), and expansion medium was replaced for 48 h with differentiation medium containing advanced DMEM/F12, 1% BSA, 100 U/ml penicillin and 100 µg/ml streptomycin, 2 mM L-glutamine, 1x N2 supplement, 1x B27 supplement (w/o vitamin A), 10 mM HEPES, 50 ng/ml hrEGF, 10 nM gastrin, 10 µM Y-27623, 2 mM sodium acetate, and 5 µM DAPT (Merck, Darmstadt, Germany).

Stainings and imaging

Proliferating cells were visualised by applying the EdU HTS Kit 488 (Sigma-Aldrich), according to manufacturer’s instructions and involving incubation with 10 µM EdU for 2 h. For dead/live analysis, 3D cultures were washed once with PBS and subsequently exposed to freshly- prepared 8 µg/ml fluorescein diacetate (FDA; Sigma-Aldrich) and 20 µg/ml propidium iodide (PI; SigmaAldrich) in PBS for 5 min at room temperature (RT), washed again with PBS and imaged immediately thereafter.
For immunostaining, monolayers were fixed apically by applying BD Cytofix fixation buffer (BD Biosciences, San Jose, CA, USA) for 10 min at RT. Cells were permeabilised with 0.2% Triton X-100 in PBS, washed and blocked for 30 min at RT with 10% goat/horse serum in PBS. Primary and secondary antibodies were diluted in 2% goat/horse serum in PBS and applied for 60 min. Washing steps were performed by applying PBS three times both apically and basolaterally for 5 min. Post-staining, one drop of Fluoroshield with DAPI (Sigma-Aldrich) was applied to each insert, and imaging was performed in situ 15 min thereafter. Antibodies used were ChgA (C-20) (Santa Cruz Biotech, Dallas, TX, USA), mCK18 (C04) (Abcam, Cambridge, UK), donkey anti-goat IgG TR (Santa Cruz Biotech), goat anti-mouse IgG FITC (Sigma-Aldrich). For Periodic Acid-Schiff (PAS) staining, cells were fixed in alcoholic formalin at RT for a minimum period of 24 h. Monolayers were stained using the PAS kit (Thermo Scientific, Waltham, MS, USA) and immediately imaged using an inverted light microscope. Fluorescence and bright field images were all obtained using the Cytation™ 5 Multi-Mode Cell Imaging Reader (BioTek Instruments Inc., Winooski, VT, USA)

Gene expression analysis

Total RNA was extracted from pelleted colonoids or monolayer cultures using the miRNeasy kit (Qiagen, Hilden, Germany), and quantified spectrophotometrically. cDNA samples were prepared in triplicate using 0.4 µg total RNA per sample, with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). PCR was performed with a Rotor-Gene 3000 (Corbett Research, Doncaster, Australia), using the QuantiTect SYBR Green PCR Kit (Qiagen), each final reaction containing 2.5 µl of cDNA, 0.3 µM of each primer, and 12.5 µl QuantiTect SYBR Green PCR Master Mix in a total volume of 25 µl. PCR conditions were 15 s at 94°C, 35-40 cycles of 30 s at 50-60°C, stopping with 30 s at 72°C. Pre-designed primers were obtained from Sigma-Aldrich. Relative gene expression was calculated using the efficiency correction method. In this method, the qPCR efficiencies and the Ct of samples vs. controls are employed to obtain the relative expression ratio of the target gene, normalized with a calibrator housekeeping gene (25). GAPDH served as calibrator housekeeping gene.

Monolayer and transepithelial electrical resistance (TEER)

Colonoids were harvested from 3D culture as described, disrupted and additionally filtered through a 40 µm cell strainer to exclude larger colonoid parts. Disrupted colonoids were applied apically in 250 µl expansion medium onto FalconTM cell culture inserts (for 24 well plate, 0.4 µM pore size, PET; Corning), pre-coated overnight with 0.67 µg/ml Purecol (Advanced BioMatrix, Carlsbad, CA, USA) at 4°C. Colonoids from one well of a 24-well culture were seeded onto one insert. Twenty four h post-seeding, expansion medium was changed to differentiation medium, and monolayers were differentiated for 48 h. TEER measurements were taken with chopstick-electrode epithelial voltohmmeter EVOM2 (World Precision Instruments, Sarasota, CA, USA). Resistance was calculated as Ω x cm² after blank values (empty insert resistance) were subtracted from total resistance readouts.

Gelatin methacrylate (GelMA) colonoid culture and bioprinting

GelMa was prepared fresh on the day of use by dissolving various concentration of GelMA (w/v) and 0.5% phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) (w/v) (both from Allevi Inc., Philadelphia, PA, USA) in PBS according to manufacturer’s instructions. For colonoid/GelMA mixture, organoids were disrupted as previously described, resuspended in a small volume of medium and mixed 1:10 with GelMA at 37°C. For manual seeding of GelMA domes, 6 µl of colonoid/GelMA mix was dispensed centrally into a black wall, clear bottom 96-well plate. Hydrogel was crosslinked using a 375 nM UV light source of 15.1 mW for 12 s at a distance of approximately 5 mm from the GelMA surface with an in-house designed crosslinking device. Medium was applied immediately after crosslinking. Colonoid/GelMA mix was loaded into a light-shielding printing cartridge and printed with the INKREDIBLE+ 3D bioprinter (Cellink AB, Gothenburg, Sweden) using a straight G-22 Luer lock needle (metal, blunt end, length of nozzle w/o needle hub 12 mm). G-code specifications were programmed to print squares in 96 well format with 3.6 x 3.6 mm size. Printing speed was set at 600 mm/min and pressure used was around 20 kPa. Printed GelMA squares were crosslinked by UV immediately postprinting, and medium was added immediately thereafter. Programmes used to generate and post process G-codes for INKREDIBLE+ application were Slic3r and Repetier-Host V1.6.1.

In-plate cryopreservation

For in-plate cryopreservation, colonoids were passaged as described and seeded as for 96-well culture. Two h post-seeding, culture medium was replaced with 100 µl Organoid-CPA (AvantiCell Science Ltd., Ayr, UK) and equilibrated for 20 min at 37°C. Plates were then gradient-frozen to a final temperature of 80°C using an EF600 gradient freezer (Asymptote Ltd., Cambridge, UK). Frozen plates were transferred to the MDF-C2156VAN-PE Cryogenic ULT Freezer (Panasonic, Kadoma, Japan) at -150°C for long-term storage. To revive colonoid cultures, culture plates were transferred to -20°C for 30 min. Subsequently, 100 µl warm unsupplemented culture medium was added to each well, and plates were placed on a heated stage at 37°C, in a static condition, for 3 min. Supernatant was removed immediately and replaced with 100 µl unsupplemented culture medium per well. Cultures were incubated at 37°C with 5% CO2, and after 20 min medium was replaced with 100 µl bovine expansion medium. Culture regime thereafter followed standard colonoid culture procedures.

Viability assay based on mitochondrial activity

Resazurin (0.25 mM in PBS; Sigma Aldrich) was added in 20 µl to cells cultured in 96-well plates. After 3 h of incubation at 37°C, supernatants were transferred to a black wall, clear bottom 96-well plate. The fluorescence of resorufin (resazurin reduction product generated by the mitochondrial activity of metabolically active cells) was determined using a Synergy™ H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments Inc.) at Ex 530/Em 590 nm. In order to assess cell proliferation, repeated viability measurements were taken on different days and proliferation was determined by calculating relative increase in mitochondrial activity in comparison to control time point.

Viability assay based on ATP production

CellTiter-Glo® 3D Cell Viability Assay (Promega, Madison, WI, USA), based on the ability of metabolically active cells to produce ATP, was performed according to manufacturer’s instructions on 96-well cultures with the additional inclusion of an ATP standard curve. Luminescence readouts were taken with the Synergy™ H4 Hybrid Multi-Mode Microplate reader.

Results

Establishment of bovine colonoid culture conditions

Bovine colon crypts were isolated in accordance with isolation protocols for mouse and human intestinal crypts (1). Upon embedding in the ECM Matrigel we found that the specified culture medium (containing Wnt-3A, hrR-spondin, hrNoggin, nicotinamide, PGE2, [Leu]15-gastrin-1, N-acteylcysteine, the TGF-βRI inhibitor A83-01, the p38 MAPK inhibitor SB202190, the GSK-3 inhibitor CHIR 99021, and the ROCK inhibitor Y-27632) supported spheroid formation from embedded crypts (Figure 2A). These culture conditions were suitable for long-term expansion of bovine colonoids as, upon splitting, new colonoids formed readily (Figure 2B) and passage numbers >30 were reached without loss of expansion potential. Expanding colonoid cultures consisted of viable spherical enteroids with apoptotic cells shedding into their lumen (Figure 2C). In addition to culture of bovine enteroids of the large intestine, the same culture conditions were found suitable for maintaining small intestinal bovine enteroid cultures (data not shown). When medium was refreshed every other day, colonoids were significantly smaller in the 5 days post-split culture compared to colonoids with daily medium refreshing (Figure 2D). However, although growth seemed to stagnate on days when colonoids had not been fed, the culture potential of colonoids was not affected as long-term culture was also achieved under the intermittent feeding regime. Upon niche factor withdrawal and additional Notch inhibition by DAPT for 48 h, the colonoid cultures differentiated, displaying a significant loss in expression of the stem cell marker Lgr5, while markers for goblet cells (Muc2), enteroendocrine cells (ChgA) and differentiated colonocytes (CA1) increased significantly (Figure 2E) (26). Moreover, a stark decrease of proliferating cells upon differentiation could be detected by EdU staining (Figure 2F).
Isolated crypts and colonoids could be cryopreserved in conventional cryovials using established enteroid suspension-cryopreservation protocols and CryoStor CS10 (Sigma-Aldrich) supplemented with 10 µM Y27623 (7, 24). Successful post-thawing colonoid culture was observed (data not shown). Novel in-plate cryopreservation of colonoids in 3D Matrigel domes is described hereafter and data presented in Figure 3.

In-plate cryopreservation of bovine colonoids and their subsequent use in cytotoxicity screenings assays

Three-dimensional cultured colonoids can be a useful tool in medium-throughput screening approaches, including drug sensitivity or cytotoxicity studies. In order to employ colonoids in such studies, labour- and time-intensive culture expansion and seeding in multi-well plates is required prior to 3D assay performance. We therefore investigated whether colonoids, previously expanded and seeded into 96 well plates, could be cryopreserved in-plate for relatively immediate post-thawing colonoid analytical applications, maintaining the same reactivity as their non-cryopreserved counterparts.
Bovine colonoids were manually seeded as 6 µl Matrigel domes in 96-well plates, and either gradientfrozen 2 h post-seeding or cultured under standard conditions. In order to achieve a uniform freezing performance within the entire Matrigel dome, it was necessary to equilibrate cells and ECM to the cryoprotecting agent prior to freezing by introducing an equilibration time of 20 min with Organoid-CPA at 37°C. Cryopreserved culture plates were sealed and stored at -150°C. After 24 h, cryopreserved plates were thawed as described in the Methods section. Further shelf life testing (time in -150°C storage) is currently under investigation. Upon thawing, Matrigel domes containing colonoids remained intact, and culture was possible in a similar fashion to non-frozen control. Both in situ freeze-thawed and control cultures were expanded for 3 days and subsequently differentiated for 48 h as previously described. Bovine colonoids, which had been cryopreserved, displayed a similar expansion efficiency to those that had not undergone the in-plate freeze/thaw procedure (Figure 3A). Upon differentiation, colonoid sensitivity to the toxic effect of staurosporine was assessed (Figure 3A). Treatment with staurosporine provoked a dose-dependent decrease in viability, measured as ATP content using the CellTiter-Glo viability assay. Control and cryopreserved cultures showed similar dose-dependent cell death in 3 independent experimental runs (Figure 3B). The average toxicity of staurosporine was not significantly different between cryopreserved and control cultures.

Bovine colonoids in 3D bioprinting applications

In view of possible use of bovine colonoids in a wider range of bio-fabrication applications, we determined whether colonoids could be used in 3D extrusion bioprinting. Several prerequisites had to be fulfilled to do so. Firstly, we needed to identify an appropriate bioink, able to support colonoid expansion. Following an initial scan of various colonoid-laden bioinks (data not shown), we determined the bioink GelMA as supportive of colonoid cultures. From a dose-response evaluation, 7.5% GelMA (w/v) was selected as the polymer concentration that less influenced the colonoid expansion potential (Figure 4A), and that supported bovine colonoid outgrowth in a similar fashion to the standard organoid ECM Matrigel (Figure 4B). These initial results were obtained from manually-seeded cultures, after which protocols for extrusion bioprinting were established.
In order to determine the suitability of GelMA EMC in 3D extrusion bioprinting, bovine colonoids were dissociated as in standard sub-culture, mixed into 7.5% GelMA supplemented with 0.1 mg/ml Matrigel, and loaded into a printing cartridge. Colonoid-containing GelMA was immediately printed as hollow 3.6 x 3.6 mm squares into 96 well cell culture plates using the INKREDIBLE+ 3D bioprinter. Using a single loaded printing cartridge, it was possible to consistently print colonoids into the inner 60 wells of a 96-well culture plate (Figure 4C) within less than 10 min. The hollow square shape was chosen to prove printing resolution. However, less complex structures, which potentially require less time to print, are expected to result in a higher throughput printing. The attempt to bioprint GelMA with even lower polymer concentrations of 5% (w/v) proved unsuccessful, as printed GelMA structures collapsed pre-crosslinking. Printed colonoid-GelMA structures were immediately UV-crosslinked with a 375 nm light source, and expansion medium was applied. One hour post-print, PI/FDA-based dead/live staining was used for assessing the viability of printed cells, and showed that printed colonoid parts were viable (Figure 4D). Dead/live staining also provided evidence of the even distribution of cell structures within the printed hydrogel, and the stability of the hydrogel shape upon crosslinking and medium addition. Analysis of bioprinted colonoid-GelMA structures 48 h post-print revealed that printed colonoids remained viable and proliferative (Figure 4E), while the printed GelMA structures remained intact.

Monolayer development on permeable inserts from 3D colonoid cultures

Although bovine colonoid cultures grown in a 3D environment are a valuable tool for in vitro studies of the mucosal epithelium, they provide only limited suitability for investigating effects of compounds and drugs applied apically (i.e., in the gut lumen). This is mainly due to the fact that the outer surface of an in vitro enteroid represents the basolateral side (i.e., the tissue side) of the epithelial monolayer in vivo, while the enteroid lumen represents the gut lumen. Therefore, monolayer cultures were obtained from cultured colonoids, grown on permeable inserts, thereby allowing for physiologically relevant testing with apical application of test compounds.
When dissociated colonoids (one well of a 24-well colonoid culture onto one insert) were seeded in expansion medium on collagen I-coated permeable inserts, cells attached to the insert within 24 h. Interestingly, inserts coated with Matrigel displayed substantial amounts of 3D outgrowth (data not shown), while collagen coated membranes displayed less 3D outgrowth and a more homogeneous monolayer formation. Monolayers were differentiated for 48 h according to the previously established 3D differentiation protocol, and differentiation of confluent monolayers was confirmed by positive staining for the epithelial marker CK18. Differentiated monolayers also included small numbers of enteroendocrine, ChgA-positive cells (Figure 5A), and of mucin-producing goblet cells (Figure 5B). Muc2 gene expression significantly increased upon monolayer differentiation while expression of stem the cell marker Lgr5 and the proliferation marker MYC significantly decreased (Figure 5C). Barrier integrity was assessed after 48 h 2 of differentiation, with a mean TEER value of 324 Ω cm (Figure 5D). Differentiated monolayers were suitable for in vitro testing, as TEER values remained at the same level for additional 24 h of culture, while treatment with the broad-spectrum kinase inhibitor staurosporine provoked a complete loss of monolayer integrity (Figure 5D).

Discussion

In this study, we describe organoid cultures, established from bovine colonic epithelium, which display culture longevity and functional flexibility. This can fulfil an industry need for better tools to model ruminant intestinal function, and support the study of agricultural bioactive agents.
We established culture conditions that support long-term culture of bovine colonoids (Figure 2). These culture conditions included the standard temperature of 37°C, based on physiological body temperature of both mice and humans. Although the resting body temperature of cows (as well as pigs) is substantially higher (39°C) (9, 27-29), it is known from porcine enteroid cultures that culture at 37°C does not significantly alter expression of differentiated enteroid markers such as Muc2 and ChgA (9). Therefore, bovine enteroid culture temperature was standardised at 37°C, thereby avoiding the need for different cell culture incubators when culturing organoids from various species and increasing feasibility for the end user. Similar to human organoid cultures, bovine 3D colonoids could be used for medium- to highthroughput cytotoxicity studies using automated seeding techniques in 384-well formats (12). As opposed to previously described primary bovine epithelial colon cultures, which readily undergo EMT (21), bovine colonoids maintain stemness over many passages. Interestingly, the culture properties of bovine small intestinal enteroids has also been the subject of a recent study. There the authors showed that bovine ileal crypts can be expanded into transcriptome- and morphology-stable enteroids over a culture period of at least 5 passages employing commercially-available murine intestinal growth medium, namely IntestiCultTM (STEMCELL Technologies, Vancouver, Canada), with additional supplementation (30). Unfortunately, the exact composition of IntestiCultTM remains undisclosed. With our data on colonic enteroids we provide additional details on the complete culture composition of bovine enteroid medium, required for stables cultures for over 30 passages.
Using colonoids in screening assays within multi-well plates not only requires an extensive laboratory skillset and equipment, but is also a labour- and time-consuming procedure. Uncoupling of 3D culture production from analytical use is therefore highly desirable. For this reason, we investigated in situ cryopreservation of pre-seeded bovine colonoid/ECM cultures in a 96-well format. While cryopreservation of cells in suspension using conventional cryovials and commercially available cryoprotectants is a routine procedure in most research laboratories, in-plate/in situ cryopreservation of 2D and 3D cell cultures is a technological approach that has received little attention so far. In general, cryopreservation of multicellular cultures displaying cell-to-cell and cell-to-matrix interactions differs significantly from that of dissociated single cell suspensions. Those interactions influence both intracellular ice formation and intercellular ice nucleation upon freezing, making cryopreservation of complex multicellular structures or tissues less feasible compared to single cell suspensions (31-33). Furthermore, in-plate cryopreservation of 3D cultures faces challenges such as post-thaw matrix retrieval. To our knowledge cryopreservation of 3D Matrigel cultures has not been reported prior to this study. We were able to show that, using a novel inplate cryopreservation technique, 3D colonoid cultures could be frozen in situ. Both outgrowth potential and sensitivity to staurosporine toxicity were not significantly different between in situ freeze-thawed and unfrozen control colonoid cultures (Figure 3). Slight variations of ATP readouts among replicate measurements can be explained by relatively uneven seeding inputs. Therefore, we expect that these fluctuations will be minimised by using automated seeding approaches and by optimising colonoid distribution in Matrigel in future developments of the system. The ability to cryopreserve complex 3D organoid/ECM assemblies in situ creates prospect of delivering such models to end-users in a convenient format that would only require limited organoid handling before analytical use. End-user friendliness is in fact a key issue in overcoming current barriers to the adoption of 3D models by industry, including the need of specialised laboratories and personnel, labour-intensity, and limited robustness (poor reproducibility) (34). Recent findings from our laboratory indicate that this cryopreservation technique can be applied to organoid models of different tissue origin, donor species and well formats (data not shown), therefore being an exciting novel technology for storing and/or delivering frozen 3D cell cultures.
Among the possible developments for bovine mucosal epithelial testing, we have described here the suitability of bovine colonoids for extrusion bioprinting applications. We have shown that colonoid bioprinting into 96 well plates is feasible with a high post-print cell viability and reproducibility. This proofof-concept opens the way to future research on the differentiation and screening potential of printed colonoids within the GelMA bioink. We expect that optimisation of the GelMA bioink will further improve the system, as it has been shown for murine enteroids that matrix stiffness influences both organoid expansion and differentiation behaviour (18). These results prove that defined 3D bioprinting of bovine colonoids into multi-well plates is a realistic prospect for enteroid and more generally organoid research, highlighting bioprinting as an alternative seeding and culture methodology, potentially enabling co-culture of various cell types with defined spatial positioning.
In this study, we have also established, from 3D colonoids, physiologically-relevant differentiated monolayers on permeable membranes that allow for apical application of test agents. On these differentiated monolayer cultures, testing can be performed over a time period of at least 24 h, as TEER values proved the integrity of the epithelium. TEER values measured in bovine monolayers were comparable to those of human enteroid-derived epithelial monolayers (7). In agreement with studies with the human system, the established bovine epithelial monolayers should be suitable not only for physiologically-relevant compound and drug testing, but they may be also used in studies of hostpathogen interactions, e.g., on bacterial adhesion and viral infection (7, 35, 36).Furthermore, we found bovine epithelial monolayers positive for the expression of cytokines such as IL-6 and IL-8 (data no shown), therefore being interesting tools for the potential study of innate immune responses of the colonic epithelium.
Taken together, our results show that it is possible to establish adult stem cell-derived bovine colonoids in 3D and monolayer cultures and apply them successfully to bioprinting and in situ cryopreservation, the first enabling reproducible assembly of 3D bovine colonoid models, and the second presenting them to end-users in a “plug and play” format. This represents the basis for developing colonoid-based models with excellent analytical performance, conferred by tissue-like cell function, which will represent an accessible, cost-effective tool for cell-based bioefficacy and biosafety testing.

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