Innervated Coculture Device to Model Peripheral Nerve-Mediated Fibroblast Activation

Abstract

Cutaneous wound healing is a complex process involving various cellular and molecular interactions, resulting in the formation of a collagen-rich scar with imperfect function and morphology. Dermal fibroblasts are crucial to successful wound healing, migrating to the wound site where they are activated to provide extracellular matrix remodeling and wound closure. Peripheral nerves have been shown to play an important role in wound healing, with loss or damage to these nerves often leading to impaired healing and the formation of chronic nonhealing wounds. Previous research has suggested that sensory nerves secrete trophic factors that can regulate wound healing, including fibroblast activation; however, the direct cell–cell interaction between nerves and fibroblasts has not been extensively studied. To address this knowledge gap, we developed an in vitro co-culture model using a device called the IFlowPlate. This model supports the long-term viability of multiple cell types while allowing for direct contact between sensory nerve cells and dermal fibroblasts. Using the IFlowPlate, we demonstrate that co-culture of dorsal root ganglia with dermal fibroblasts increases fibroblast proliferation, collagen and α-smooth muscle actin expression, and secretion of pro-wound healing factors, suggesting that nerves can promote wound healing by modulating fibroblast activation. The IFlowPlate offers a user-friendly and high-throughput platform to study the in vitro interactions between nerves and a variety of cell types that can be applied to wound healing and other important biological processes.

Introduction

Wound healing involves a series of coordinated processes in which cellular, molecular, and biochemical phenomena dynamically interact to reconstitute damaged tissues in response to injury.¹⁻⁵ In most adult mammals, the result is a collagen-rich scar that fails to replicate the initial tissue phenotype and function.¹ Cutaneous scars exhibit reduced elasticity, electrophysiological conductivity, and diversity in extracellular matrix (ECM) composition, and increased mechanical stiffness, culminating in dysregulation of normal tissue functioning.²⁻⁹ During cutaneous wound healing, connective tissue-derived fibroblasts migrate into the wound and undergo activation to remodel the ECM, replacing it with a dense collagen matrix that forms the fibrotic scar.¹⁰⁻¹⁶ Activated fibroblasts, also called myofibroblasts, are characterized by a morphological change from spindle to stellate shape, expression of α-smooth muscle actin (αSMA), and an increase in cell proliferation, migration, and collagen I expression.^17,18 In the skin, myofibroblasts remodel the ECM after injury via the expression of matricellular proteins and matrix metalloproteinases (MMPs) and aid in wound closure by altering the contractile properties of the dermis.^2−5,10,11 Fibroblast activation is known to be induced by inflammatory signals (e.g., interleukins), contact signals (e.g., Notch and integrins), and binding of receptor tyrosine kinase ligands such as fibroblast growth factors (FGFs), transforming growth factor (TGF)β, and platelet-derived growth factor (PDGF).^17,19 While fibroblast activation is a necessary part of wound healing, it requires tight regulation both spatially and temporally to avoid excessive scarring and other pathological outcomes.

Most tissues of the body are either in proximity to or directly innervated by peripheral nerves,²⁰ and it has been shown in both non-mammalian and mammalian models of wound healing and regeneration that nerves have an important role in the response to tissue damage.²¹⁻³⁹ Loss or damage of peripheral nerves can disrupt or even ablate wound closure and scar deposition, resulting in the formation of chronic nonhealing wounds.^40,41 In the skin, experimental depletion of innervation prior to injury impairs the onset of inflammation,⁴² decreases wound closure rate,⁴³ and increases the wound surface area.^43,44 Previous work has demonstrated that sensory nerves can regulate the wound healing process through the secretion of trophic factors such as FGFs.^45,46 Studies examining the role of nerves in skin wound healing mainly employ animal models to investigate tissue-level dynamics with little known about the direct cell–cell interactions between nerves and dermal fibroblasts. In vitro models can be used to examine the interaction of specific cell types, genes, and proteins at a molecular level, allowing for a better understanding of how each player participates in the response to damage.^{15,19,32,33,47} In particular, co-culture models provide the opportunity to examine the interactions between two or more key cell types involved in wound healing.⁴⁸ One strategy to investigate contact-independent mechanisms is the use of conditioned media. Cells or tissue can be cultured, and the spent media collected and introduced to a different cell type to observe the effect of the donor secretome on the recipient.⁴⁹⁻⁵² Alternatively, direct co-culture of different cell types enables the combined study of contact-dependent and independent mechanisms; however, it can be difficult to accurately model the cell–cell configurations found in vivo. Despite the importance of peripheral nerves in many biological processes, most in vitro models do not include innervation as a component of the culture system.

To provide an innervated model that compartmentalizes cell types while allowing specific cellular interactions, we developed an in vitro co-culture model of sensory nerves and dermal fibroblasts using a commercially available culture device, the IFlowPlate384.⁵³⁻⁵⁵ This user-friendly device allows for the compartmentalization of cell types, requires a relatively small quantity of cells and media, and has high-throughput capabilities. With the IFlowPlate, we demonstrate that primary mouse dorsal root ganglia (DRG) can be cultured with robust neurite sprouting and maintenance of nerve viability for at least 21 days. Furthermore, co-culture of dermal fibroblasts with DRG in the IFlowPlate promotes fibroblast activation, as evidenced by increased fibroblast proliferation, collagen I and α-smooth muscle actin expression, and secretion of pro-wound healing factors. These proof-of-concept data suggest that the pro-healing properties of peripheral nerves are executed in part through modulation of fibroblast activation. Our findings present the first use of the IFlowPlate as a model to interrogate nerve–cell interactions in a high-throughput manner that can be used to introduce innervation as a variable in a range of downstream biological applications.

Materials and Methods

Cell Culture

All cells were cultured at 37 °C and 5% CO2. The normal human dermal fibroblast (HDF) cell line was purchased (VWR; 10175-138), and cells were seeded at 1 × 10⁴ cells/mL and cultured in Fibroblast-2 media containing fibroblast basal medium (VWR, 10172-066), 1 ng/mL fibroblast growth factor (FGF) (VWR, 10771-944), 5 mg/mL insulin (4 mg/mL) (Thermo Fisher, 12585014), 2% fetal bovine serum (FBS), and 1%(v/v) ABAM (Wisent, 50-115-EL). Half-media changes were performed every 2 days, and cells were cultured until 70–90% confluent. Cells from passages five or six were used for all experiments. Primary mouse dorsal root ganglia (DRG) were cultured in complete Neurobasal media containing Neurobasal A medium (Invitrogen, 21103049), 2% (v/v) W-21 (Wisent, 003-018-XL), 1% (v/v) GlutaMAX (Thermo Fisher, 35050061), 1% (v/v) Pen/Strep (Wisent 450-201-EL), and 1.25 ng/mL NGF (VWR 10781-146).

Dorsal Root Ganglia Harvest

Experimental procedures were performed in accordance with the Canadian Council on Animal Care University of Guelph’s Guide to the Care and Use of Experimental Animals and approved by the Animal Care Committee at the University of Guelph (Animal Use Protocol #4746). Eight- to 12-week-old female C57BL/6 mice were purchased from Charles River and housed in groups on a standard 12:12 h light/dark cycle with food and water provided ad libitum. To obtain DRG, mice were euthanized in CO2, decapitated, and the spinal column was removed and rinsed in chilled PBS. Identical long incisions were made on each lateral edge of the spine along the ventral length, allowing a small strip to be removed to expose the spinal cord (Figure 1A). A midsagittal bisection was made through the spine, the spinal cord was removed, and up to 35 DRG were collected from each animal from the cervical, thoracic, and lumbar region using fine forceps (Fisher Scientific, 16100121) and placed in chilled PBS. Excess myelin was excised using a #3 scalpel (Fine Science Tools, 1003-12; blade: Feather Safety Razor, 15 08-916-5D) under an Olympus SZ-6145TR trinocular stereo microscope before DRGs were further cleaned using a digestion cocktail of 1.0 mL of Dispase II (MilliporeSigma, SCM133) and 0.5 mL of TrypLE (Gibco, 12605028) for 30 min at 37 °C and 5% CO2 in a 1.5 mL tube. Once the digestion process was complete, 0.5 mL of the digestion cocktail was removed from the tube before the remaining 1 mL, along with the digested DRGs, was placed into a prepared Petri dish with 2 mL of room-temperature PBS.

Figure 1.

A schematic diagram of the harvest, seeding, and co-culture setup of DRG-HDF in the IFlowPlate. (A) DRGs are harvested, excess myelin is removed, and the samples are digested before seeding into the IFlowPlate. (B) (i) Seeding of DRG. (1) One DRG is seeded in chamber A of a triplet. The top-down view below shows the recommended location of seeding to minimize the distance for neurite growth through the channel. (2) Immediately after DRG seeding, 20 μL of Neurobasal media (pink) is added to each chamber, from left to right. (3) 24 h later, 60 μL of Neurobasal is added to each chamber, from left to right. Half-media maintenance changes are performed by removing 40 μL from all chambers and adding 40 μL of fresh Neurobasal media to each. (ii) Seeding of HDFs. (1) DRG after 14 days of culture. Steps 2 and 3 are performed in quick succession. (2) Medium is removed from chamber B. (3) HDFs are seeded into chamber B in 80 μL of Fibroblast-2 media. (iii) Coculture medium changes. All steps are performed in quick succession. (1) 40 μL of medium is removed from all chambers. (2) 40 μL of Fibroblast-2 media is added to chamber B. (3) 40 μL of Neurobasal media is added to chambers A and C.

Dorsal Root Ganglia Seeding and Culture Maintenance

To seed DRG in the IFlowPlate device, all wells in the IFlowPlate384 (OrganoBiotech, A001) were coated with 20 μL per well of 20 μg/mL Poly-d-Lysine (PDL, Sigma, P6407-5 mg) for 2 h at 37 °C, or overnight at 4 °C. The PDL was removed, chambers were rinsed with DPBS, and 20 μL of 10 μg/mL laminin (Corning, CB-40232-1 mg) was added to each chamber and incubated overnight at 37 °C. Immediately before DRG seeding, laminin was removed, and chambers were rinsed with DPBS and allowed to dry. Up to 30 DRGs were seeded in chamber A of each triplet (Figure 1Bi). DRGs were placed at the channel entrance using a 25G needle. 20 μL of complete Neurobasal media was added to each chamber, and the plates were incubated at 37 °C and 5% CO2 for 16 to 24 h to promote DRG adherence and viability while preventing lifting from the surface, before adding an additional 60 μL of media for a total of 80 μL in each chamber (Figure 1Bi). For DRG culture maintenance, half-media changes were performed every second day.

For DRG-conditioned media experiments, glass coverslips were placed in the wells of 6-well or 48-well plates. Coverslips were coated with 1 mL (in a 6-well plate) or 125 μL (in a 48-well plate) of 20 μg/mL Poly-d-Lysine (PDL, Sigma, cat. no. P6407-5 mg) for 2 h at 37 °C, or overnight at 4 °C. The PDL was removed, followed by rinsing with DPBS and then 1 mL (in a 6-well plate) or 125 μL (in a 48-well plate) of 10 μg/mL laminin (Corning, CB-40232-1 mg) and incubated overnight at 37 °C. At the time of DRG seeding, laminin was removed from culture plates, washed once with DPBS, and allowed to dry. Individual DRG were seeded in 20 μL of complete Neurobasal media in a droplet using fine forceps to allow adherence to the coverslip without floating away and left for 16 to 24 h at 37 °C and 5% CO₂. Into each well was added 2 mL (in a 6-well plate) or 500 μL (in a 48-well plate) of complete Neurobasal media. Half-media changes were performed every second day.

IFlowPlate Coculture

Following 14 days of DRG culture to allow for maximal neurite extension, media were removed from chamber B while keeping Neurobasal media in the DRG-containing chamber A, and HDFs were seeded in 80 μL of Fibroblast-2 media at a density of 1.25 × 10³ cells per chamber (Figure 1Bii). For HDF-alone controls, all three chambers in a triplet were seeded with HDFs in the absence of DRG. To label HDFs for imaging of the IFlowPlate, HDFs were labeled with CellTracker CM-DiI (Invitrogen, C7000) according to the manufacturer’s directions and incubated for 24 h prior to passaging and seeding into the IFlowPlate. For co-culture triplets, a final volume of 80 μL of Neurobasal media was placed in chambers A and C, and 80 μL of Fibroblast-2 media was placed into chamber B. HDF alone control triplets received 80 μL of Fibroblast-2 media in all three chambers. Half-media changes were performed every 2 days by removing 40 μL of media from all wells and adding in 40 μL of Fibroblast-2 media to chamber B, followed by rapid addition of 40 μL of Neurobasal media to chambers A and C (Figure 1Biii).

Fibroblast Culture in Dorsal Root Ganglia Conditioned Media

Half of the total media was removed from cultured DRG after 14 days of culture, snap-frozen in liquid nitrogen, and stored at −80 °C. This collection was repeated three times, and collected media were pooled for the conditioned media experiments. HDFs were first seeded in a 48-well plate at a density of 7.5 × 10³ cells/well in Fibroblast-2 media and cultured for 24 h to allow for acclimation. Media were then exchanged with one of the following treatments: CTRL (3:1 Fibroblast-2: unconditioned Neurobasal media) or DRG-CM (3:1 Fibroblast-2: DRG-conditioned Neurobasal media). Dilution of DRG-CM with fresh Fibroblast-2 medium in a 3:1 ratio was used to support HDF viability and avoid the use of a high proportion of spent media. Cells were cultured for 7 days in treatment media with half-media changes performed every 2 days before fixation.

Staining and Immunocytochemistry

Cultures were maintained for 7 days, and then cells were fixed with 4% paraformaldehyde (PFA) for 20 min and washed with DPBS three times for 5 min each. Blocking solution (0.1% v/v Triton-X-100 (VWR, 97063-864) and 5% w/v (mg/μL) bovine serum albumin (Wisent, 800-095-EG) in DPBS) were added to cells at a volume of 20 μL for 1 h at room temperature. The blocking solution was removed and replaced with 20 μL of primary antibody diluted in blocking solution and incubated overnight at 4 °C. Primary antibodies and their dilutions are as follows: mouse anti-TUBB3 (1:300, Proteintech, 66375-1-Ig), rabbit anticollagen I (1:1000, Abcam, ab138492), rat anti-Ki67 (1:250, Invitrogen, 14-5698-82), and mouse anti-α-SMA (1:500, Cell Signaling, 48983S). After 24 h, the primary antibody solution was removed, cells were washed with DPBS three times for 5 min each, and then cells were treated with 20 μL of secondary antibody diluted in blocking solution or rhodamine phalloidin (1:300, Invitrogen, R415) and DAPI (Invitrogen, D3571) for 2 h at room temperature in the dark. Secondary antibodies are as follows and were used at a 1:500 concentration unless otherwise stated: AlexaFluor488 goat antimouse (Invitrogen, A-21121), AlexaFluor488 goat antirat (Invitrogen, A11006), AlexaFluor546 goat antimouse (Invitrogen, A11003), and AlexaFluor647 goat antirabbit (Invitrogen, A21244). The secondary solution was removed, cells were washed with DPBS three times for 5 min each, and then wells were filled with DPBS for imaging.

Image Acquisition and Analysis

Samples were imaged using a confocal microscope (Olympus, FV1200) at 10× magnification. One image was taken per IFlowPlate chamber, and z-stacks were acquired with a thickness of 8.5 μm per slice. Images were analyzed and quantified using Fiji ImageJ software (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, Maryland). Z-stack images were processed using the Z-Project function (Image > Stacks > Z-Project, set to Sum Slices) and then converted to 16-bit format (Image > Type >16-bit). To quantify the collagen I and α-SMA signals, a threshold was manually determined, which accurately captured images with both the highest and lowest pixel intensity, and this threshold was applied to all images within the same experimental group. A region of interest (ROI) was then created using the Create Selection function (Edit > Selection > Create Selection), followed by using the Measure function (Analyze > Measure) to quantify the total number of positive pixels. The total number of collagen I- or α-SMA-positive pixels was normalized to the number of DAPI-positive cells in each image.

For the DAPI and Ki67 images, a threshold was set for each image, which allowed clear resolution of individual nuclei while minimizing background signal. DAPI-positive nuclei were automatically counted using the Analyze Particles function (Analyze > Analyze Particles, size between 50–1000 μm², circularity between 0.20–1.00, show set to Overlay, summarize and overlay boxes ticked), and any nuclei not included by running this function were manually counted using the Cell Counter function (Plugins > Analyze > Cell Counter > Cell Counter). DAPI and Ki67 channels were then merged, and costaining nuclei were manually counted using the Cell Counter function to obtain the proportion of Ki67+ and DAPI+ cells over a total number of DAPI+ cells.

The morphology of HDFs was measured as previously described in ImageJ.⁵⁶ The phalloidin channel from each image was isolated and overlaid with DAPI, and five cells per image were selected and manually outlined using the Polygon selection. Cell area, perimeter, and circularity were calculated and averaged across three or more images per experimental group and three or more biological replicates.

Cytokine Array

To identify secreted factors from HDFs co-cultured with DRG in the IFlowPlate, 40 μL of media was removed from each well containing HDFs after 7 days of co-culture and pooled over 1 week (sampling conducted three times total) and kept at 4 °C for short-term storage (<6 days). For long-term storage, samples were snap-frozen in liquid nitrogen and stored at −80 °C. Prior to analysis, all samples were thawed and centrifuged at 1000g for 5 min at room temperature to remove debris and obtain aliquots of 1 mL. Collected media from HDFs co-cultured with DRG or HDFs only were analyzed using a commercially available Human Angiogenesis and Growth Factor 17-Plex Discovery Assay Tissue-Cell Culture (MilliporeSigma, Burlington, MA). The multiplexing analysis was performed using the Luminex 200 system (Luminex, Austin, TX) at Eve Technologies Corp. (Calgary, Alberta, Canada). Base-supplemented fibroblast medium was used as a negative control. Luminex xMAP technology was used for multiplexed quantification of the 17 human cytokines, chemokines, and growth factors. Seventeen markers were simultaneously measured in the samples according to the manufacturer’s protocol. Assay sensitivities of these markers range from 0.2–42.8 pg/mL for the 17-plex. Individual analyte sensitivity values are available in the MilliporeSigma MILLIPLEX MAP protocol.

Statistical Analysis

Statistical analysis and graphing were both performed with Prism GraphPad software (GraphPad Software, Boston, MA). IFlowPlate experiments were analyzed using Student’s t test. Conditioned media experiments were analyzed using one-way ANOVA, and all ANOVA were followed by multiple comparisons posthoc tests. Grubbs’ outlier tests were performed for all data sets, and any identified outliers were removed from the final analyses. Sample sizes are indicated in each figure legend, and a minimum of three biological replicates were performed for each experiment, unless stated otherwise. Data are shown as mean ± standard deviation unless otherwise noted.

Results and Discussion

The IFlowPlate Supports Dorsal Root Ganglia Viability and Neurite Extension

The IFlowPlate is a commercially available culture device consisting of triplicate wells connected by channels that have been previously shown to support the formation of perfused, vascularized colorectal cancer, liver, kidney, placenta, and lung organoids.^{53−55,57−59} For the first time, we adapted the IFlowPlate for the culture of primary mouse DRG to develop an innervated co-culture model. After seeding one DRG within a chamber of the IFlowPlate triplet and 2 weeks of culture, we achieved an extension of βIII-tubulin positive neurites from the DRG chamber, through the channel, and into the adjacent HDF chamber (Figure 2). DRG viability was also maintained for at least 21 days in the culture. We observed both that the initial DRG placement was important in the growth of neurites through the channel, with DRG in closer proximity exhibiting neurite extension through the length of the channel, whereas DRG seeded farther from the channel opening did not sprout neurites that entered the channel, and that there was variability between DRG in success of neurite sprouting (Supporting Information Figure 1A–C). We also tested both polystyrene and pressure-sensitive adhesive sheet bottom IFlowPlates and found that successful DRG neurite sprouting (i.e., sprouting from the DRG body into the channel) occurred more frequently on the polystyrene surface. On average, we found that approximately 55% of DRG sprout neurites into the connecting channel on the polystyrene surface IFlowPlate compared to 35% on the pressure-sensitive adhesive bottom (Supporting Information Figure 1D). Neurite extension through the channel length and at least partial infiltration into the HDF chamber was achieved by 2 weeks, which is consistent in timing with similar reports of cultured DRG neurite extension.⁶⁰ Our system also included laminin coating to support DRG sprouting; however, to promote directed neurite extension, future work could use soluble or patterned chemoattractant gradients or other biochemical cues in the channel.^61,62 Additionally, we observed the presence of DAPI-positive cells in the channel, likely from the DRG body. The use of whole primary DRG is advantageous as it more closely mimics the in situ microenvironment; however, DRGs are composed of multiple cell types, including neurons, Schwann cells, and satellite glial cells, which can play different roles in the modulation of the tissue repair response⁶³⁻⁶⁵ and may delaminate from the main DRG body during culture. Overall, we demonstrate that the IFlowPlate can be used to support the viability and compartmentalized neurite extension of primary DRG to create an innervated culture model.

Figure 2.

Representative composite fluorescent image of co-cultured dorsal root ganglion (DRG) and human dermal fibroblasts (HDF) in the IFlowPlate. DRG were seeded in the left chamber, followed by seeding of CellTracker-labeled HDF (red) in the center chamber. After 7 days of co-culture, chambers were fixed, and immunocytochemistry was performed for βIII-tubulin (green) and DAPI (blue). βIII-tubulin-positive neurites from DRG extend through the connecting channel and into the HDF-containing chamber. Scale bar: 400 μm.

Coculture of Dermal Fibroblasts with Dorsal Root Ganglia Promotes Fibroblast Activation

To demonstrate the application of our co-culture model and determine whether peripheral nerves can modulate dermal fibroblast activation, we co-cultured DRG with human dermal fibroblasts (HDFs) in the IFlowPlate and analyzed HDF activation after 7 days using immunocytochemistry (Figure 3A). An advantage of the IFlowPlate is that it supports the compartmentalization of more than one cell type with different media compositions; by following the sequential steps in Figure 1B, cells can be seeded in their respective chambers with minimal mixing of media through the connecting channel. Maintenance of an equal volume in all chambers minimizes flow between chambers and enables the compartmentalization of different cell types during seeding. During the co-culture period following initial cell seeding, it is likely that some diffusion of medium between chambers occurs, but it does not impact cell viability of either cell type. The minimal mixing during cell seeding was confirmed visually by adding colored water to a triplicate of chambers and observing the mixing of dyes over 60 min. We mimicked the cell seeding protocol and imaged the chambers immediately after the media change and 5 and 60 min later, finding minimal mixing of the dyes over time (Supporting Information Figure 2).

Figure 3.

Direct co-culture of HDFs with DRG promotes HDF activation, which is not replicated with DRG-conditioned medium alone. (A) Schematic of experimental timeline for IFlowPlate co-culture. (B) Representative images of staining for nuclei (DAPI, blue), proliferation (Ki67, green), and collagen I (yellow) in co-cultured HDFs. DRG co-culture increases (C) HDF proliferation, (D) HDF collagen I expression, and (E) HDF αSMA expression. (F) Representative images of staining for nuclei (DAPI, blue) and αSMA (red). (G) Representative images of staining for nuclei (DAPI, blue) and phalloidin (red). DRG co-culture increases (H) average HDF area and (I) perimeter. (J) Schematic of experimental timeline for the DRG-conditioned media experiment. (K) Representative images of staining for nuclei (DAPI, blue), proliferation (Ki67, green), and collagen I (yellow) in HDFs cultured in DRG-conditioned medium (DRG-CM) or naïve fibroblast media (CNTRL). Quantification of the (L) average percentage of proliferating cells and (M) average number of collagen I pixels per cell. Data expressed as mean ± SD, n = 4–7 biological replicates, unpaired Student’s t test. Scale bar = 100 μm.

Next, we investigated fibroblast activation and determined that, compared to HDF culture alone, co-culture of HDFs with DRG resulted in significantly increased HDF proliferation, average collagen I expression, and average αSMA expression (Figure 3B–F). Proliferation and collagen I expression are hallmarks of fibroblast activation and important in wound closure and matrix remodeling during wound healing,^{2−5,10,11,17,19} and the expression of αSMA is considered a marker of activated fibroblasts.^66,67 We also observed a significant increase in the average area and perimeter of HDFs with DRG co-culture, which suggests there is a change in morphology, further indicative of HDF activation (Figure 3G–I). Importantly, significant fibroblast activation was only observed with IFlowPlate co-culture; when HDFs were cultured with media harvested from cultured DRG (i.e., DRG-conditioned media) (Figure 3J), there was no statistically significant change in HDF proliferation or collagen I expression after 7 days of culture (Figure 3K–M). These results demonstrate that coculture of HDFs with DRG in the IFlowPlate promotes HDF activation, which culture in DRG-conditioned media alone is insufficient to replicate.

The most well-characterized method by which peripheral nerves regulate wound healing and regeneration is the secretion of soluble factors such as growth factors, neuropeptides, and cytokines,^{17,22,27,29−31,33,35,45,68} which may support fibroblast activation. A previous study which co-cultured DRG with skin explants reported an increase in dermal fibroblast proliferation and differentiation into myofibroblasts, which was hypothesized to involve nerve-derived signaling molecules such as vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), and substance P.⁴⁶ In the skin, peripheral nerve endings are in close proximity to cells of the dermis and epidermis;²⁴ therefore, it is possible that the IFlowPlate configuration induces HDF activation because it more faithfully replicates the association of neurites with HDFs, which is absent with the use of conditioned media alone. Alternatively, there may be a threshold of exposure duration or concentration of nerve-derived factors needed to increase HDF activation that is achieved only by the continuous exposure of HDF to DRG in the IFlowPlate. Indeed, it has been reported in animal models of regeneration that a minimum threshold of innervation is required to elicit an effect on the regenerative process.^{35,39,40,46,69} Our results demonstrate that with our innervated co-culture model, we are able to elicit significant activation of HDFs, which cannot be replicated with the culture of HDFs in DRG-conditioned media alone.

Co-Culture with DRG Promotes Expression of Pro-Healing Factors in Fibroblasts

While other co-culture devices can support neurite extension through channels or similar configurations,^70,71 the ability to conduct high-throughput assays is often lacking. The IFlowPlate is advantageous because the small chamber area and volume require low numbers of both cells and media, reducing reagent use and cost, and can also be used to culture up to 128 replicates in one plate. Our DRG harvest protocol can yield up to 35 DRG from one mouse; therefore, one IFlowPlate device can support three to four biological replicates (i.e., mice) simultaneously with multiple experimental groups within each replicate. To test the high-throughput capabilities of the IFlowPlate in our innervated model, we co-cultured DRG with HDFs for 7 days and compared the HDF secretome with and without DRG. Using one IFlowPlate device, we were able to simultaneously seed enough DRG to perform four biological replicates (i.e., mice) with at least 12 technical replicates per animal (Figure 4A). We sampled media from the HDF chamber to analyze changes in the HDF secretome with and without DRG co-culture. Each chamber holds 80 μL of media, and 40 μL is removed with each media change for collection and analysis; therefore, seeding 12 chambers allowed us to collect a sufficient media volume and protein concentration for cytokine analysis with one sampling. Furthermore, the open top configuration of the IFlowPlate allows for quick and easy sampling of media with minimal disruption to the co-culture system.

Figure 4.

IFlowPlate co-culture with DRG increases the expression of trophic factors by HDF. (A) Schematic of experimental design. DRG from four separate mice (A–D) were seeded into one IFlowPlate device, and HDFs were seeded into the adjacent chamber along with control chambers of HDF alone (“control”). DRG-HDFs were cultured for 7 days, and then media from HDF chambers were collected for cytokine array analysis. (B) Heatmap of log-transformed protein concentration for each biological replicate of blank media (CNTRL), HDF culture alone, and HDF-DRG. A yellow box indicates a fold change of at least 2 compared to HDF. (C) Fold change in detected protein concentration in DRG-HDF co-culture to HDF alone. N = 4 mice; ∼12 DRG pooled per mouse. No statistical significance.

To determine if co-culture with DRG alters trophic factor secretion by HDFs, we used a commercially available cytokine array for angiogenic factors. To account for the potential diffusion of soluble factors between the DRG and HDF chambers over time, a human-specific kit was used to detect only HDF-derived cytokines. The re-establishment of the microvascular network following injury to the dermis is an important step in the formation of granulation tissue and overall wound healing.^72,73 Dermal fibroblasts help to directly promote angiogenesis after injury through the release of factors such as FGF-1 and FGF-2^74,75 and indirectly via matricellular proteins, which can alter the dynamics of soluble signaling molecules such as bone morphogenetic protein-2 (BMP-2) and FGFs.^2,10,11,47 Chronic or impaired wound healing is often coupled with impaired angiogenesis, with reduced levels of the growth factors that promote blood vessel formation.⁷⁶ Previous work using human gingival fibroblast-conditioned media found that culturing fibroblasts, keratinocytes, or endothelial cells in this media increased migration and proliferation and promoted wound healing through modulation of inflammation and angiogenesis.⁵¹ Additionally, others have reported that conditioned media derived from senescent dermal fibroblasts increased the fibrotic response of treated dermal fibroblasts,⁵² demonstrating that fibroblasts produce soluble factors that can alter the wound healing response.

We found detectable levels of all cytokines tested for in HDF-conditioned media, with or without DRG co-culture, compared to the blank medium control (Figure 4B; Supporting Information Table 1). Comparing the fold change in protein concentration of HDF-DRG co-culture to HDFs alone, we observed that co-culture with DRG increased the protein concentration of epidermal growth factor (EGF) (6.8-fold), heparin-binding epidermal growth factor-like growth factor (HB-EGF) (3.0-fold), hepatocyte growth factor (HGF) (2.0-fold), and bone morphogenic protein-9 (BMP-9) (2.0-fold) although not to a statistically significant level (Figure 4C). EGF is a mitogen for both endothelial cells and dermal fibroblasts and is secreted by fibroblasts.^77,78 It can also act in a paracrine function on keratinocytes to promote re-epithelialization^77,78 and has been shown to accelerate wound closure in diabetic mice.⁷⁹ A member of the EGF family, HB-EGF can be found bound to the ECM or in a soluble form when released via proteolytic cleavage. It is a major component of postwounding fluid⁸⁰ and promotes keratinocyte migration during skin wound healing in a mouse model.⁸¹ When delivered locally with VEGF-A and PDGF-BB, HB-EGF also improves wound healing in a diabetic mouse model.⁸² HGF is a growth factor that regulates cell growth, motility, and other functions in a variety of cell types and is reported to be expressed by dermal fibroblasts.⁸³ As with the other factors found elevated in our HDF-DRG culture, HGF accelerates wound healing in diabetic rats,⁸⁴ and also has a role in matrix remodeling of the wound by increasing MMP1 expression in dermal fibroblasts.⁸⁵ Lastly, BMP-9 is a growth factor known for its role in osteogenesis, but it is also important for the wound healing response in skin. BMP-9 levels are decreased in diabetic patients, and it promotes re-epithelialization⁸⁶ and induction of collagen I expression in fibroblasts in mouse models of skin wounds.⁸⁷

Taken together, our results demonstrate that with the IFlowPlate we can simultaneously culture at least 48 individual DRGs from up to four individual mice within one device and perform a secretome analysis. We found that in culture HDFs secrete trophic factors involved in angiogenesis and wound healing, some of which moderately increase in concentration in the presence of DRG. The elevation of these cytokine levels in HDFs co-cultured with DRG suggests that nerves may indirectly modulate important wound healing processes such as re-epithelialization, matrix remodeling, and angiogenesis through their control of fibroblast activation and trophic factor secretion.

Conclusions

We present a simple and versatile co-culture platform with high-throughput capability to study the direct signaling of nerves with dermal fibroblasts. Our proof-of-concept results show that the IFlowPlate device can support seeding and viability of DRG, neurite growth, and co-culture with HDFs for at least 21 days. The design of the IFlowPlate supports the simultaneous culture and testing of a large quantity of innervated samples under experimental conditions. It also facilitates standard end-point analyses such as immunocytochemistry as well as repeated sampling of the chamber media for high-throughput analyses such as the cytokine array. Future work can take advantage of our innervated co-culture system to create 3D hydrogel-based tissues that will also be compatible with robotic liquid handling systems for automation of culture maintenance.

Using our co-culture platform, we determined that the presence of nerves promotes HDF activation and secretion of pro-healing factors, which aligns with previous work in models of nerve loss or damage in wound healing.^25,40 These data contribute to our knowledge of the role of sensory neurons in fibroblast activation and suggest that peripheral reinnervation after injury may promote fibroblast activation to drive subsequent wound healing. By understanding the interactions between nerves and fibroblasts and how they contribute to the healing response, we can gain a greater understanding of the mechanisms underlying scar formation and wound healing that can help direct development of therapeutics to promote regeneration over scarring. Lastly, this proof-of-concept study demonstrates the ability to introduce innervation as a variable in the IFlowPlate to study the interactions of nerves with other cell types and improves our ability to accurately model in vivo tissues.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsbiomaterials.4c01482.

• Supporting Information Figure 1: Representative composite images of DRG variation in sprouting in the IFlowPlate. Figure 2: Visualization of solution transfer between chambers of the IFlowPlate triplets during HDF seeding. Table 1: Raw detected protein concentration (pg/mL) of conditioned media (PDF)

Author Contributions

^∥ S.C. and A.S. are co-first authorship. S.C.: Formal analysis, investigation, methodology, and writing—original draft. A.S.: Investigation, Methodology, Writing—review and editing, and visualization. K.B.: Formal analysis, investigation, and methodology. S.K.: Methodology. B.Z.: Conceptualization, resources, and writing—review and editing. S.L.P.: Conceptualization, project administration, funding acquisition, supervision, writing—review and editing, and visualization.

The authors declare the following competing financial interest(s): B.Z. holds equities in OrganoBiotech, Inc., which is commercializing the IFlowPlate platform used in this work.