Name: 3D Flipwell Engineering for Developing Asynchronous Systems for Toxicologic and Immunomodulatory Therapies in Bacterial, Gut, and Immune Cells (Video) | JoVE Journal
Uploaded: 2025-10-17T12:40:00.000+00:00
Duration: 11 min
Description: Learn to engineer a 3D Flipwell co-culture system for studying gut mucosal interactions and drug responses. This method enables evaluation of bacterial, epithelial, and immune cell crosstalk.

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5 minutes

The primary author expresses sincere gratitude to Dr. Saori Furuta for her superb mentorship skills, continuing guidance and support across long distances, and skillful and continuous project design that foster fantastic learning opportunities. The authors also thank Dr. Vandana Sharma for microbiology expertise and guidance and Dr. Veani R. Fernando for the preparation of THP-1 macrophages, Gebremichal Gebretsadik Weidengus and Syed Islam for their help with the preparation of the necessary media, cell and bacterial cultures prior to filming.
The authors also thank Metro Health Medical Center/Division of Cancer Biology for their hospitality and support during the filming process. The primary author thanks the University of Michigan Dr. Erin Janssen, Dr. Eliza Tsou and Caroline Foster for the project support in the form back up supplies and the XL lab coat used during filming.
Additionally, the authors thank Drs. Andrew Kleinhenz and William T. Gunning at the Electron Microscopy Facility, and Cassandra Zamora and Kristin Kirschbaum at the Instrumentation Center at the University of Toledo for constructive suggestions for EM imaging. This work was supported by the startup fund from University of Toledo Health Science Campus, College of Medicine and Life Sciences, Department of Cancer Biology to S.F; Ohio Cancer Re-search Grant (Project #: 5017) to S.F; Medical Research Society (Toledo Foundation, #206298) Award to S.F; American Cancer Society Research Scholar Grant (RSG-18-238-01-CSM) to S.F; and National Cancer Institute Research Grant (R01CA248304) to SF.

<ol><li>Goldsmith, J. R., Sartor, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+diet+on+intestinal+microbiota+metabolism%3A+downstream+impacts+on+host+immune+function+and+health%2C+and+therapeutic+implications%5BTitle%5D)+AND+%22J+Gastroenterol%22%5BJournal%5D)">The role of diet on intestinal microbiota metabolism: downstream impacts on host immune function and health, and therapeutic implications.</a> J Gastroenterol. 49 (5), 785-798 (2014).</li>
<li>Xuan, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microbial+dysbiosis+is+associated+with+human+breast+cancer%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Microbial dysbiosis is associated with human breast cancer.</a> PLoS One. 9 (1), e83744(2014).</li>
<li>Yoo, J. Y., Groer, M., Dutra, S. V. O., Sarkar, A., McSkimming, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gut+Microbiota+and+Immune+System+Interactions%5BTitle%5D)+AND+%22Microorganisms%22%5BJournal%5D)">Gut Microbiota and Immune System Interactions.</a> Microorganisms. 8 (10), 1587(2020).</li>
<li>Kim, H. J., Li, H., Collins, J. J., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contributions+of+microbiome+and+mechanical+deformation+to+intestinal+bacterial+overgrowth+and+inflammation+in+a+human+gut-on-a-chip%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip.</a> Proc Natl Acad Sci U S A. 113 (1), E7-E15 (2016).</li>
<li>Donkers, J. M., Eslami Amirabadi, H., van de Steeg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intestine-on-a-chip%3A+Next+level+in+vitro+research+model+of+the+human+intestine%5BTitle%5D)+AND+%22Curr+Opinion+Toxicol%22%5BJournal%5D)">Intestine-on-a-chip: Next level in vitro research model of the human intestine.</a> Curr Opinion Toxicol. 25, 6-14 (2021).</li>
<li>Noel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+primary+human+macrophage-enteroid+co-culture+model+to+investigate+mucosal+gut+physiology+and+host-pathogen+interactions%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">A primary human macrophage-enteroid co-culture model to investigate mucosal gut physiology and host-pathogen interactions.</a> Sci Rep. 7, 45270(2017).</li>
<li>Beamer, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+3D+Flipwell+system+that+models+gut+mucosal+microenvironment+for+studying+interactions+between+gut+microbiota%2C+epithelia+and+immunity%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Novel 3D Flipwell system that models gut mucosal microenvironment for studying interactions between gut microbiota, epithelia and immunity.</a> Sci Rep. 13 (1), 870(2023).</li>
<li>Beamer, M. A., Furuta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Redefining+Cell+Culture+Using+a+3D+Flipwell+Co-culture+System%3A+A+Mimetic+for+Gut+Architecture+and+Dynamics+In+Vitro%5BTitle%5D)+AND+%22Curr+Protoc%22%5BJournal%5D)">Redefining Cell Culture Using a 3D Flipwell Co-culture System: A Mimetic for Gut Architecture and Dynamics In Vitro.</a> Curr Protoc. 5 (2), e70107(2025).</li>
<li>Fernando, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reprogramming+of+breast+tumor-associated+macrophages+with+modulation+of+arginine+metabolism%5BTitle%5D)+AND+%22Life+Sci+Alliance%22%5BJournal%5D)">Reprogramming of breast tumor-associated macrophages with modulation of arginine metabolism.</a> Life Sci Alliance. 7 (11), e202302339(2024).</li>
<li>Sharma, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunogenic+shift+of+arginine+metabolism+triggers+systemic+metabolic+and+immunological+reprogramming+to+suppress+HER2+%2B+breast+cancer%5BTitle%5D)+AND+%22Cancer+Metab%22%5BJournal%5D)">Immunogenic shift of arginine metabolism triggers systemic metabolic and immunological reprogramming to suppress HER2 + breast cancer.</a> Cancer Metab. 13 (1), 15(2025).</li>
<li>Zheng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Correction+of+arginine+metabolism+with+sepiapterin-the+precursor+of+nitric+oxide+synthase+cofactor+BH%284%29-induces+immunostimulatory-shift+of+breast+cancer%5BTitle%5D)+AND+%22Biochem+Pharmacol%22%5BJournal%5D)">Correction of arginine metabolism with sepiapterin-the precursor of nitric oxide synthase cofactor BH(4)-induces immunostimulatory-shift of breast cancer.</a> Biochem Pharmacol. 176, 113887(2020).</li>
<li>Ren, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reduced+Basal+Nitric+Oxide+Production+Induces+Precancerous+Mammary+Lesions+via+ERBB2+and+TGF%CE%B2%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Reduced Basal Nitric Oxide Production Induces Precancerous Mammary Lesions via ERBB2 and TGFβ.</a> Sci Rep. 9 (1), 6688(2019).</li>
<li>Trejo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Elasticity+and+wrinkled+morphology+of+Bacillus+subtilis+pellicles%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Elasticity and wrinkled morphology of Bacillus subtilis pellicles.</a> Proc Natl Acad Sci U S A. 110 (6), 2011-2016 (2013).</li>
<li>Sadaghian Sadabad, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+coculture+system+shows+mutualism+between+anaerobic+faecalibacteria+and+epithelial+Caco-2+cells%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">A simple coculture system shows mutualism between anaerobic faecalibacteria and epithelial Caco-2 cells.</a> Sci Rep. 5, 17906(2015).</li>
<li>Umehara, Y., Aoyagi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+simple+cultured+cell-anaerobic+microbial+co-culture+system+using+liquid+paraffin%5BTitle%5D)+AND+%22J+Biosci+Bioeng%22%5BJournal%5D)">Development of a simple cultured cell-anaerobic microbial co-culture system using liquid paraffin.</a> J Biosci Bioeng. 135 (6), 487-492 (2023).</li>
</ol>

The authors declare no conflict of interest. The authors hereby disclaim all warranties, whether expressed or implied, to the fullest extent permitted by law. Under no circumstances shall the authors be held liable, whether under any statute, contract, tort, or any other legal theory, for any special, incidental, indirect, punitive, multiple, or consequential damages arising from or related to the use of tools in constructing the Flipwells, the use of BSL reagents, cell lines, bacterial cultures, the use of Flipwell system under anaerobic environment in an anaerobic chamber. This limitation of liability includes, but is not limited to, any unspecified manner of Flipwell usage not explicitly described herein.

To recreate gut mucosal architecture in vitro, we have designed a simple co-culture system that comprises stratified layers of gut bacteria, mucus-producing colon epithelial cells, and macrophages. This multi-co-culture system has allowed us to study the effects of crosstalk between the bacterial secretome, gut epithelia, and macrophages on inducing immunogenic reprogramming and mucosal defense mechanisms.
One of the important cautions for creating 3D Flipwells is that the assembly mu...

Immune cell-gut microbiome crosstalk greatly influences the efficacy of different disease treatments. Such a crosstalk is markedly modified by external stimuli, including but not limited to host metabolic abnormalities, dietary changes, disease manifestation, and pharmacologic treatment. These changes could lead to an imbalance in the microflora composition, a condition termed dysbiosis1,2,3.
To better understand the interactions between microbes and host immune cells in the gut, elaborate co-culture systems have been developed by different groups in an attempt to recapitulate the gut mucosal environment. From the gut-on-chip system developed by Kim et al.4 to a more complex assembly using a specialized absorbent substrate5, these co-culture systems are not simple to build and are unable to retain the full spectrum of secreted components due to their absorption by the co-culture substrate. Noel et al. thus developed an alternative co-culture system to model the gut mucosa that is relatively easier to fabricate6. This new system utilizes a single culture insert with colon epithelial cells seeded on one side of the membrane and pre-differentiated macrophages on the other side6. However, seeding pre-differentiated macrophages requires their pre-differentiation somewhere else and their dissociation and replating into a co-culture, potentially affecting their cellular integrity and viability.
By incorporating improvements to these previous systems, we developed a novel three-dimensional (3D) multi-layered co-culture system that we termed the 3D Flipwell system7,8. This system is composed of cell culture inserts stacked back-to-back, allowing for the synchronous progression of colon epithelial polarization, mucus formation, macrophage differentiation, and polarization within a single co-culture. This system enables us to examine changes in the gut mucosal environment and interactions between gut bacteria, epithelial cells, and immune cells. Additionally, this system can be used to examine the effects of external stimuli, including bacterial metabolites, toxins, dietary nutrients, and pharmaceuticals, on the gut mucosa and the rest of the body7,8.
By utilizing the novel co-culture insert system, we examined the responses of different cellular components to sepiapterin (SEP), the endogenous precursor of BH4 (tetrahydrobiopterin)-a cofactor of nitric oxide synthase (NOS). We previously showed that SEP exerts pro-immunogenic activities on the breast tumor microenvironment by reprogramming tumor-associated macrophages to a pro-immunogenic type9,10,11,12. We demonstrated that SEP induces synchronous events of biofilm formation by gut bacteria, mucus production by colon epithelium, and pro-immunogenic polarization of macrophages in co-cultures, indicating activation of a mucosal defense mechanism7. These findings support the utility of this new co-culture system in modeling the gut mucosal microenvironment7.
The overall goal of this protocol is to provide step-by-step instructions for the construction of this new co-culture of gut bacteria, gut epithelia, mucus, and immune cells to model the gut mucosal environment. We also demonstrate how we could visualize the crosstalk of co-cultured cells under the influence of an external stimulus, for example, SEP treatment. Further research utilizing this co-culture system is needed to determine secreted cellular components that facilitate the interactions between different cells in co-culture, as well as to test the utility of this co-culture model for high-throughput screening of new therapeutics. Furthermore, to attain the physiological condition of the gut mucosal environment, this co-culture system would require further modifications. For example, to co-culture aerobic mammalian cells with anaerobic bacteria, these bacteria would need to be separately cultured in anaerobic media that remove oxygen or overlain with liquid paraffin that prevents oxygen entry, as reported elsewhere. Such advanced utility and application of this co-culture system would warrant further investigation.

<table><tbody><tr><td>0.4% PFA (see recipe in Reagents and Solutions)</td><td>described in text</td><td></td><td></td></tr><tr><td>10-ml serological pipette</td><td>Corning</td><td>4488</td><td></td></tr><tr><td>12 well TC Plate,&amp;nbsp; Costar</td><td>Corning</td><td>3513</td><td></td></tr><tr><td>15-ml conical tubes, SpectraTube</td><td>Alkali Scientific</td><td>C2715</td><td></td></tr><tr><td>200 &amp;micro;l tips&amp;nbsp;</td><td>USA Scientific</td><td>1111-1700</td><td></td></tr><tr><td>24-well inserts - Culture Inserts for 24-well plates, PET 0.4 &amp;micro;m&amp;nbsp;</td><td>BrandTech Scientific</td><td>782710</td><td></td></tr><tr><td>3D Flipwells pre-made, UV sterilized&amp;nbsp;</td><td>Make in lab</td><td></td><td></td></tr><tr><td>4% PFA (see recipe in Reagents and Solutions)</td><td>described in text</td><td></td><td></td></tr><tr><td>50 mM Sepiapterin stock (SEP, See recipe in Reagents and Solution)</td><td>Career Henan Chemical Co</td><td>CAS#: 17094-01-8</td><td></td></tr><tr><td>50-ml conical tubes</td><td>Greiner Bio-One</td><td>227261</td><td></td></tr><tr><td>5-ml serological pipette</td><td>Corning</td><td>4487</td><td></td></tr><tr><td>70-95% Ethanol (Ethyl Alcohol)</td><td>Decon Labs</td><td>04-355-151</td><td></td></tr><tr><td>Aluminum foil</td><td>Fisher Scientific</td><td>01-213-100</td><td></td></tr><tr><td>Animal Feeding needles, 20 gauge 1.5&amp;rdquo; length, single use&amp;nbsp;</td><td>Fisher Scientific</td><td>01-208-87</td><td></td></tr><tr><td>Antibody Dilution Buffer (see recipe in Reagents and Solutions)</td><td>described in text</td><td></td><td></td></tr><tr><td>Attofluor Cell Chamber&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>A7816</td><td></td></tr><tr><td>Autoclave tape</td><td>Fisher Scientific</td><td>15-901-111</td><td></td></tr><tr><td>Bacillus subtilis wt (3A1T) strains in glycerol stock ()</td><td>Bacillus Genetic Stock Center (https:// bgsc. org/</td><td></td><td></td></tr><tr><td>BioGrip Pink Nitrile powder free latex free gloves</td><td>Top Quality Manufacturing</td><td>P2810</td><td></td></tr><tr><td>Blocking Buffer (see recipe in Reagents and Solutions - 10% Normal Goat Serum in PBS)</td><td>described in text</td><td></td><td></td></tr><tr><td>Blocking Buffer (see recipe in Reagents and Solutions)</td><td>described in text</td><td></td><td></td></tr><tr><td>Brightfield microscope (Nikon or equivalent) Eclipse Ts2</td><td>Nikon</td><td>TS2</td><td></td></tr><tr><td>Caco-2 cells</td><td>ATCC</td><td>HTB-37</td><td></td></tr><tr><td>Cell culture incubator, 37&amp;deg;C, 5% CO2 (Eppendorf CellXpert)</td><td>Eppendorf</td><td>6734010015</td><td></td></tr><tr><td>Centrifuge (Eppendorf, Centrifuge 5804/5804 R or equivalent</td><td>Eppendorf</td><td>22622501</td><td></td></tr><tr><td>Centrifuge tubes 50 ml SpectraTube</td><td>Alkali Scientific</td><td>C2750</td><td></td></tr><tr><td>Culture inserts for 12-well plate, PET, 0.4 &amp;micro;m, sterile</td><td>BrandTech Scientific, BRAND Insert 2in1, BIO-CERT</td><td>782730</td><td></td></tr><tr><td>DAPI VectaShield mounting medium</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>Deep petri dish bottoms, 100 x 25 mm Deep Polystyrene Stackable Petri Dish, sterile&amp;nbsp;</td><td>USA Scientific</td><td>8609-0625</td><td></td></tr><tr><td>Drummond Pipet Aid XP</td><td>Drummond&amp;nbsp;</td><td>4-000-101</td><td></td></tr><tr><td>Eppendorf 5804 Centrifuge with a swing bucket rotor for 50 ml conical tubes</td><td>Eppendorf</td><td>22622501</td><td></td></tr><tr><td>Forceps, stainless steel, sterile autoclaved, 2x sets</td><td>VWR</td><td>82027-438</td><td></td></tr><tr><td>Forceps, stainless steel, sterile autoclaved, 2x sets, Specimen Forceps</td><td>VWR</td><td>82027-438</td><td></td></tr><tr><td>Glass flask 500 ml</td><td>Corning&amp;nbsp;</td><td>4980-500</td><td></td></tr><tr><td>Hemacytometer (Neubauer with cover slips)</td><td>Thermo Fisher Scientific</td><td>50-312-90</td><td></td></tr><tr><td>HT-29-MTX-E12 cells</td><td>Millipore Sigma</td><td>12040401</td><td></td></tr><tr><td>ImmunoPen (optional)</td><td>Fisher Scientific</td><td>4021761EA</td><td></td></tr><tr><td>Kevlar-based or other cut-resistant gloves (optional)</td><td>Hardware store or Amazon</td><td></td><td></td></tr><tr><td>Lab tape or shipping tape</td><td>Fisher Scientific&amp;nbsp;</td><td>159015R</td><td></td></tr><tr><td>Laminar Flow hood 1300 Series A2 BSC Class II</td><td>ThermoFisher Scientific</td><td>1375</td><td></td></tr><tr><td>Laminar flow hood with UV light SterilGARD</td><td>The Baker Company</td><td>SG403A-HE</td><td></td></tr><tr><td>Markers solvent proof</td><td>VWR</td><td>52877-310 or 95042-566</td><td></td></tr><tr><td>Media for Caco-2 cells (DMEM High Glucose-based, see recipe in Reagents and Solutions)</td><td>described in&amp;nbsp; text</td><td></td><td></td></tr><tr><td>Media for HT-29 cells (DMEM High Glucose, see recipe in Reagents and Solutions)</td><td>described in text</td><td></td><td></td></tr><tr><td>Media for THP-1 cells (RPMI, see recipe in Reagents and Solutions)</td><td>described in text</td><td></td><td></td></tr><tr><td>Microcentrifuge tube, 1.7 ml, sterile&amp;nbsp;</td><td>Alkali Scientific</td><td>C3017-ST</td><td></td></tr><tr><td>Microcentrifuge tubes</td><td>Thermo Fisher</td><td>3451</td><td></td></tr><tr><td>Miller LB media (see recipe in Reagents and Solutions)</td><td>Sigma-Aldrich</td><td>&amp;nbsp;L3522</td><td></td></tr><tr><td>Needle nose pliers, 2x sets</td><td>Hardware store or Amazon</td><td></td><td></td></tr><tr><td>Nitrile powder free latex free gloves Aurelia Amazing</td><td>MidSci</td><td>92885</td><td></td></tr><tr><td>Petri dish lids, sterile Falcon Petri Dish sterile 100x15 mm style</td><td>Corning Inc</td><td>351029</td><td></td></tr><tr><td>Phorbol myristate acetate (PMA) (100 ng/&amp;mu;l, see recipe in Reagents and Solutions)&amp;nbsp;</td><td>InvivoGen</td><td>Tlrl-pma</td><td></td></tr><tr><td>Pipet tips (10 &amp;micro;l, 200 &amp;micro;l, 1000 &amp;micro;l) PurePoint or equivalent</td><td>Alkali Scientific&amp;nbsp;</td><td>ST1010-CS, ST1200-CS, ST1000-CS</td><td></td></tr><tr><td>Plastic wrap</td><td>Grocery store</td><td></td><td></td></tr><tr><td>Purecol working collagenase solution (100 &amp;micro;g/mL)</td><td>Advanced BioMatrix</td><td>5005-100ML</td><td></td></tr><tr><td>Round cover slips #1 1 oz (Corning Float Glass or Fisherbrand)</td><td>Corning or Fisherbrand</td><td>50143822 or 12-546-2 25CIR-2</td><td></td></tr><tr><td>Scalpel blades, sterile, disposable&amp;nbsp;</td><td>Mopec or Feather #10</td><td>AJ136 or 2975#10</td><td></td></tr><tr><td>Sepiapterin (SEP, 100 &amp;micro;M, see recipe in Reagents and Solution)</td><td>Career Henan Chemical Co</td><td>CAS#: 17094-01-8</td><td></td></tr><tr><td>Shaking incubator set for 220 rpm 37&amp;deg;C</td><td>Eppendorf New Brunswick Scientific</td><td>M12990080</td><td></td></tr><tr><td>Silicone Adhesive Loctite Clear Silicone IDH or 3M Marine Grade Silicone Sealant Clear with a nosetip</td><td>Loctite or 3M</td><td>1745658 or PN08019</td><td></td></tr><tr><td>Single channel pipet Eppendorf Research Plus, 200 &amp;micro;l</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Single channel pipettes (10 &amp;micro;l, 200 &amp;micro;l, 1000 &amp;micro;l)</td><td>Eppendorf</td><td>2231001122</td><td></td></tr><tr><td>Spectrophotometer NanoDropOneC</td><td>Thermo Fisher Scientific</td><td>13-400-519</td><td></td></tr><tr><td>Sterile deionized water&amp;nbsp;&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Sterile phosphate buffered serine (PBS) without calcium or magnesium</td><td>Fisher Scientific</td><td>&amp;nbsp;BSS-PBS-1X6</td><td></td></tr><tr><td>Syringe 3 ml or 5 ml with Luer-Lok Tip</td><td>BD</td><td>309585 or 309603</td><td></td></tr><tr><td>THP-1 cells</td><td>ATCC</td><td>TIB-202</td><td></td></tr><tr><td>Transfer pipets, sterile</td><td>Fisherbrand</td><td>13-711-20</td><td></td></tr><tr><td>Triton X-100</td><td>Millipore Sigma</td><td>X100</td><td></td></tr><tr><td>Trypan blue solution</td><td>VWR</td><td>97063-702</td><td></td></tr><tr><td>Trypsin-EDTA 0.25%</td><td>Thermo Fisher Scientific</td><td>25200-056</td><td></td></tr><tr><td>Wipes Kimwipes Professional with LintGuard Anti-Stat Polyshield</td><td>Kimberly-Clark</td><td>34120</td><td></td></tr><tr><td>&lt;strong&gt;Primary Antibodies:&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-human CD163</td><td>Abcam</td><td>156769</td><td></td></tr><tr><td>Anti-human CD68</td><td>Novus Biologicals</td><td>NB100-683-0.1 mg</td><td></td></tr><tr><td>Anti-human CD80, E3Q9V</td><td>Cell Signaling Technologies</td><td>15416</td><td></td></tr><tr><td>Anti-human CK20&amp;nbsp;</td><td>Life Technologies</td><td>PA5-82875</td><td></td></tr><tr><td>Anti-human MUC2 (996/1)</td><td>ThermoFisher</td><td>MA5-12345</td><td></td></tr><tr><td>&lt;strong&gt;Secondary Antibodies:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor 488 Goat anti-Rabbit IgG (for CD80 and CK20)</td><td>ThermoFisher</td><td>A11008</td><td></td></tr><tr><td>Alexa Fluor 594 Goat anti-Mouse IgG (for MUC2 and CD68 or CD163)</td><td>ThermoFisher</td><td>A11005</td><td></td></tr><tr><td>&lt;strong&gt;Reagents and Solutions:&amp;nbsp;&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Caco-2 Medium:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM with high glucose (4.5g/L) and 1% (v:v) GlutaMax&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>35050061</td><td></td></tr><tr><td>FBS 10% (v:v) heat-inactivated</td><td>Sigma</td><td>F2242-500ML</td><td></td></tr><tr><td>Non-essential amino acids 1% for Caco-2 media</td><td>Thermo Fisher Scientific</td><td>11140076</td><td></td></tr><tr><td>Pen Strep 1% (v:v)</td><td>Sigma</td><td>F2242-500ML</td><td></td></tr><tr><td>&lt;strong&gt;HT-29-MTX Medium&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM with high glucose (4.5g/L) and 1% (v:v) GlutaMax&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>35050061</td><td></td></tr><tr><td>FBS 10% (v:v) heat-inactivated</td><td>Sigma</td><td>F2242-500ML</td><td></td></tr><tr><td>HEPES (10 mM final concentration)</td><td>Thermo Fisher Scientific</td><td>15630080</td><td></td></tr><tr><td>Pen Strep 1% (v:v)</td><td>Sigma</td><td>F2242-500ML</td><td></td></tr><tr><td>&lt;strong&gt;THP-1 Medium:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>FBS (10% v:v) heat-inactivated fetal bovine serum</td><td>Sigma&amp;nbsp;</td><td>F2242-500ML</td><td></td></tr><tr><td>Glucose (4.5 g/L final concentration)</td><td>Thermo Fisher Scientific</td><td>A249001</td><td></td></tr><tr><td>GlutaMax 1% (v:v)&amp;nbsp; 2 mM final concentration</td><td>Thermo Fisher Scientific</td><td>35050061</td><td></td></tr><tr><td>HEPES (10 mM final concentration)</td><td>Thermo Fisher Scientific</td><td>15630080</td><td></td></tr><tr><td>Pen Strep 1% (v:v)</td><td>Sigma</td><td>F2242-500ML</td><td></td></tr><tr><td>Roswell Park Memorial Institute (RPMI)-1640 medium</td><td>Thermo Fisher Scientific</td><td>11875119</td><td></td></tr><tr><td>Sodium Pyruvate (1 mM final concentration)</td><td>Thermo Fisher Scientific</td><td>11360070</td><td></td></tr><tr><td>&lt;strong&gt;Purecol working collagenase solution (dilution to 1:30 100 &amp;micro;g/ml):&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Purecol 3 mg/ml 1:30 to 100 &amp;micro;g/ml&amp;nbsp;</td><td>Advanced BioMatrix</td><td>5005-100ML</td><td></td></tr><tr><td>sterile deionized water</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;L-Sepiapterin stock solution (50 mM):&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>L-Sepiapterin (SEP)</td><td>Career Henan Chemical Co</td><td>CAS#: 17094-01-8</td><td></td></tr><tr><td>&lt;strong&gt;Phorbol myristate acetate stock solution (PMA, 5 mg/mL)&lt;/strong&gt;</td><td>InvivoGen</td><td>Tlrl-pma</td><td></td></tr><tr><td>BSA 1% Bovine serum albumin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>5470</td><td></td></tr><tr><td>DMSO&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>D2650</td><td></td></tr><tr><td>Miller LB</td><td>Sigma-Aldrich</td><td>&amp;nbsp;L3522</td><td></td></tr><tr><td>Normal Serum from the same species as the secondary antibody</td><td>Sigma&amp;nbsp;</td><td>NS02L-1ML</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>441244</td><td></td></tr></tbody></table>

3d flipwell engineering for developing asynchronous systems for toxicologic and immunomodulatory therapies in bacterial, gut, and immune cells

3D Flipwell Engineering for Developing Asynchronous Systems for Toxicologic and Immunomodulatory Therapies in Bacterial, Gut, and Immune Cells

To study the roles of bacterial secretome in immune cell polarization and gut mucosal homeostasis, we developed a 3-dimensional co-culture model using commercially available inserts in a back-to-back stacked format. We termed this system 3D Flipwells, and successfully co-cultured bacteria, colon epithelial, and immune cells in stratified layers to recapitulate the gut mucosal microenvironment. This co-culture system enables the seeding of colon epithelial cells on one side of the membrane and undifferentiated non-adherent monocytes on the other, allowing for the synchronous progression of colon epithelial polarization, mucus formation, and monocyte-macrophage differentiation and polarization within a single co-culture without the need for detaching and replating pre-differentiated macrophages. This system is easy to construct and only requires a few readily available materials and tools. We tested its utility by analyzing the responses of co-culture components to sepiapterin (SEP), the endogenous precursor of BH4 (tetrahydrobiopterin) -- a cofactor of nitric oxide synthase (NOS). We demonstrated that SEP treatment of 3D Flipwell induced biofilm formation by gut bacteria, mucus production by colon epithelium, and pro-immunogenic polarization of macrophages. This indicated that SEP triggered activation of a mucosal defense mechanism through crosstalk between different cellular components. Further experiments are needed to investigate the role of mucosal communications that lead to immune cell reprogramming and to evaluate the utility of this co-culture system to screen for novel immunomodulatory therapies.

To determine the biological relevance of Flipwell co-cultures of gut bacteria, colon epithelia, and macrophages, we treated the co-cultures with sepiapterin (SEP), a modulator of arginine metabolism, to promote the production of nitric oxide9,10,11 in comparison to the control DMSO. We determined the effects of SEP treatment on each component of co-cultures using immunofluorescent (IF) microscopy and scanning electron microscopy...

This article provides a detailed protocol with key steps to guide the 3D Flipwell engineering and utilization. We describe and show how to assemble the co-culture insert stacks and utilize them for co-culturing stratified layers of gut bacteria, gut epithelia, and macrophages to model the gut mucosal environment.

To study the roles of bacterial secretome in immune cell polarization and gut mucosal homeostasis, we developed a 3-dimensional co-culture model using ...

We have developed a 3D co-culture system that mimics the gut mucosal environment to study the serial crosstalk and to evaluate the drug responses which could be utilized for drug screening and for understanding mucosal cellular interactions. Different groups have developed several different culture system to model the gut mucosal environment by using very complex bioengineering techniques. But instead, we developed a co-culture system which is much easier to make using less expensive materials.Our co-culture system revealed that a proteinogenic drug triggers coordinated responses among gut bacteria, epithelial and immune cells, activating host immunity likely with our bacterial metabolites and mediating cross-species communication. We aim to study both aerobic and anerobic bacterial strains and their metabolites to uncover immune modulating effects, paving the way for new immunotherapeutic strategies based on bacterial metabolites. To begin, open a Petri dish cover and set it aside.Place a scalpel blade on the edge of the Petri dish bottom. Take a set of sterile tweezers and gently grab the insert bottom up to remove it from the packaging. With the other hand, take the sterile scalpel blade and bring it up to the insert.With a C-shaped motion, pierce the membrane at the edge and gently slide the scalpel blade around the insert bottom, staying close to the plastic wall. Cut the PET membrane out and use the second set of tweezers to remove it. Then use the scalpel blade to scrape any white membrane shavings to clean the edge and rim of the insert.Next, gently spread a 2-3 millimeter bead of silicone all the way around the insert bottom, being careful not to touch the membrane. Pick up the second set of sterile forceps and carefully lift the first insert without the membrane out of its protective pack. Bring the two inserts together bottom to bottom so that the bottom rims align.When the Flipwell stack is glued, place it inside the original sterile pack or a deep Petri dish to dry for 72 hours. Use the lid from the specified Petri dishes to cover the stack assembly. To sterilize the stack assemblies, use sterile forceps to hang the Flipwell stack on the rim of the specified deep Petri dish rim.Then, close the glass sash of the biosafety cabinet and turn on the ultraviolet light. To test for leakage prior to collagen coating, first add 500 microliters of sterile PBS or sterile deionized water. The next day, aspirate the liquid used for testing before drying the stacks for 1-2 hours inside the biosafety cabinet.To coat the membrane with collagen, open the Petri dish lid and let the Flipwell hang off the Petri dish rim. Carefully add 200 microliters of collagen solution to one side of the insert stack. Aspirate the collagen solution after one hour, then pipette 200 microliters of sterile PBS After aspirating the PBS, cover the Petri dish with a lid and let the membrane dry for 60 minutes inside the cabinet.When the membrane is dry, use sterile forceps or tweezers and flip the Flipwell to the opposite side and let it hang off the Petri dish rim. To make the bacterial insert, place two sets of sterile forceps and the 24-well inserts inside the biosafety cabinet. With sterile tweezers, lift the insert from its sterile pack.With the second set of tweezers or forceps, break off the small plastic feet at the bottom of the insert. Test the 24-well insert by fitting it inside one of the sterile Flipwell co-culture insert stacks or the original 12-well inserts. To begin seeding the Flipwells, seed 500 microliters of the epithelial cells lines on each side of the Flipwell's apical side.Cover the assemblies with a Petri dish lid, then incubate at 37 degrees Celsius with 5%carbon dioxide overnight until the cells attach. Open the Petri dish lid and aspirate the DMEM media from the apical side of the Flipwell stack. With sterile forceps, pick up the Flipwell carefully by the hook and rotate it by 180 degrees.With the second set of sterile forceps, grab the Flipwell stack by the other hook now facing upward. Then lower the assembly back into the Petri dish and hang the hook over the Petri dish rim. Add 500 microliters of THP-1 cell suspension to the apical side of the co-culture insert stack.Adjust the HT-29 media for the colon epithelial cells by adding media to the deep well Petri dish. To remove any air bubbles, hold the Flipwell stack by the arm with sterile forceps. Carefully lower a gavage needle in and under the insert assembly and very gently place the soft tip up to the air bubble.Carefully and very slowly pull the syringe plunger up and watch the air bubble disappear slowly. Then carefully remove the needle and syringe from the Flipwell. After the air bubbles have been removed with a gavage needle, culture both cell types in the cell culture incubator.With forceps, set the insert inside a sterile Petri dish and cover with the lid. Transfer the Petri dish with the Flipwells into the biosafety cabinet and set aside on the opposite side of the dish with bacterial inserts. Pipette out 300 microliters of the DMEM media from the apical side of the Flipwell to allow for the bacterial insert, then cover with the Petri dish lid.Now, add 50 to 100 microliters of Bacillus subtilis culture to the 24-well bacterial inserts and insert it into the Flipwell top side. Rotate the arm and hang it from the rim of the Flipwell co-culture insert stack. Hold the stack with the second set of sterile forceps.After a three-hour incubation, use sterile forceps to remove the bacterial insert from the Flipwell. Place the Flipwell inside a 50-milliliter conical tube without disassembly. To disassemble the Flipwell for electron microscopy, hold it with both hands and twist off each glued part of the stack.Rotate the insert with the membrane and set it down With the membrane facing up. Hold the insert, carefully cut out the membrane with a scalpel blade, then place it inside a 1.7-milliliter tube containing paraform aldehyde solution. For confocal microscopy, add 300 microliters of sterile PBS to the apical side.Carefully remove the stack from the Petri dish after pipetting out the PBS. Now, flip the Flipwell and add 300 microliters of PBS to the now facing upward RPMI side of the Flipwell co-culture insert stack, then aspirate the phosphate buffered saline. Using a 200-microliter pipette, create a large droplet of PBS.Twist off the insert assembly to separate into the inserts. Then carefully position the Flipwell stack with THP-1 cells on top of the 200-microliter drop of PBS. Add 200 microliters of permeabilization buffer to the top side with colon epithelial cells and leave for 10 minutes.For immunofluorescent staining, pipette 300 microliters of primary antibody to the top of the insert. Add 300 microliters of primary antibody to the well of a 12-well plate, then place the insert inside the well on top of the droplet. Cover the antibody well.Staining showed dramatic increases of mucus secretion in the gut epithelial compartment after sepiapterin treatment, as indicated by the increases of the epithelial marker protein CK20 and mucosal protein MUC2. In THP-1 monocultures, sepiapterin treatment significantly increased the expression of the M1 macrophage marker CD80, while down modulating M2 macrophage marker CD163, indicating M1 polarization. Scanning electron microscopy revealed increased mucus secretion from epithelial cells in sep treated co-cultures.Sepiapterin treatment induced macrophage morphology resembling M1 phenotype in both monocultures and co-cultures. Bacterial cells in sepiapterin-treated monocultures displayed wrinkled surfaces characteristic of biofilm formation.

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Learn to engineer a 3D Flipwell co-culture system for studying gut mucosal interactions and drug responses. This method enables evaluation of bacterial, epithelial, and immune cell crosstalk.

3D Flipwell Engineering for Developing Asynchronous Systems for Toxicologic and Immunomodulatory Therapies in Bacterial, Gut, and Immune Cells (Video) | JoVE Journal

3D Flipwell Engineering for Developing Asynchronous Systems for Toxicologic and Immunomodulatory Therapies in Bacterial, Gut, and Immune Cells

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