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

Summary

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.

Abstract

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.

Introduction

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 dysbiosis^1,2,3.

To better understand the interactions between microbes and host immune cells in the gut, elaborate co-culture systems have been developed by different groups ....

Protocol

1. 3D Flipwell engineering

1. All work should be done inside a cell culture cabinet. Place all supplies inside the cabinet.
2. Open the Petri dish cover and set aside. Open the scalpel blade from its protective casing and place it on the edge of the Petri dish bottom. Ensure sterility.
   CAUTION: Careful handling of the scalpel blade is required. Use cut resistant, fiber reinforced gloves to protect hands if needed.
3. Open the sterile protective packaging of the 12-well commercial insert. Ensure sterility. Take a set of sterile tweezers. Gently grab the insert and remove it from the packaging.
4. Hold the insert so ....

Results

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 oxide^9,10,11 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.......

Discussion

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.......

Materials

List of materials used in this article

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