This research was primarily supported by funding from the National Institutes of Health (UG3/UH3TR002136, UG3/UH3TR003090). In addition, we thank Dr. Paul Manner (University of Washington) for providing the human tissue samples and Dr. Jian Tan for their help in isolating the MSCs and creating the cell pool.

The following protocol follows the ethical guidelines of the University of Pittsburgh and the human research ethics committee of the University of Pittsburgh. Information on the materials used in this study is listed in the Table of Materials.
1. Manufacturing 3D-printed bioreactors
<ol>
	<li>Use a computer software to design osteochondral (Figure 2A) and miniJoint bioreactors (Figure 2B) that include chambers, inserts, and lids. The dimension information for each part is shown in Figure S1.</li>
	<li>Transfer the design to a 3D printer, and print with a photopolymer ink.</li>
	<li>Rinse the 3D-printed parts (Figure 2C-F) with 15 mL of 95% ethanol three times. Then, fully crosslink the printed pieces for 200 s in a flashlight polymerization device.</li>
	<li>Add O-rings to inserts and lids (Figure 2C, D), and test if the parts fit (Figure 2G, H).</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64186/64186fig02.jpg" /> 
Figure 2: Fabrication of the different components to make the miniJoint bioreactor. (A,B), 3D models of bioreactors for creating (A) osteochondral and (B) miniJoint chips. (C,D) 3D printed (C) lids and (D)&#160;inserts with the O-ring installed. (E,F) 3D printed chambers for (E) osteochondral and (F) miniJoint tissue culture. (G,H) Assembly of (G) osteochondral and (H) miniJoint chips. <a href="https://www.jove.com/files/ftp_upload/64186/64186fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Engineering the tissue components
NOTE: The processes for the fabrication of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and methacrylated gelatin (GelMA) are described in previous studies8,9.
<ol>
	<li>To create GelMA, follow the steps below.
	<ol>
		<li>Add 17 g of gelatin Type B to 500 mL of distilled water, and then mix on a shaker for 30 min at 37 &#176;C.</li>
		<li>Then, add 13 mL of methacrylic anhydride, place back on the 37 &#176;C shaker, and allow to shake overnight.</li>
		<li>The following day, aliquot the GelMA into individual dialysis bags, with ~60 mL in each bag.</li>
		<li>Place all the dialysis bags in distilled water with a stir bar, and allow for 7 days of dialysis. Change the water multiple times per day, and leave the bags at 4 &#176;C overnight.</li>
		<li>On day 7, freeze the GelMA at &#8722;80 &#176;C. Once completely frozen, proceed to lyophilization.</li>
		<li>Place the GelMA in a dish in the vacuum chamber of a lyophilizer, and allow for freeze-drying. Ensure the GelMA is completely dried prior to removal from the lyophilizer.</li>
	</ol>
	</li>
	<li>Dissolve the GelMA in Hank&#39;s balanced salt solution (HBSS with Ca2+ and Mg2+) at 15% (w/v). To ensure the pH is at 7.4, add NaOH in small amounts until the pH reaches 7.4. Supplement the solution with 1x antibiotic-antimycotic and 0.15% (w/v) LAP based on the volume acquired. Store the 15% GelMA solution at &#8722;20 &#176;C until use, and protect it from light.</li>
	<li>Place 3D-printed dual-flow bioreactor chambers, lids, and inserts into autoclave bags, and autoclave at 121 &#176;C for 20 min with steam and then for 20 min with dry heat.</li>
	<li>Inside the biological safety cabinet, soak the bioreactor chambers, lids, and inserts in 15 mL of sterile phosphate-buffered saline overnight, after which allow them to dry.</li>
	<li>Isolate human bone marrow-derived MSCs from total joint arthroplasty surgical waste with IRB approval (University of Pittsburgh and University of Washington).
	<ol>
		<li>Specifically, flush out the bone marrow from the trabecular bone of the femoral neck and head, and resuspend it in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM).</li>
		<li>Filter the suspension through a 40 &#181;m strainer, and centrifuge the flow-through at 300 x g for 5 min.</li>
		<li>Remove the supernatant, resuspend the pellets using growth medium [DMEM, 10% fetal bovine serum (FBS), and 1x antibiotic-antimycotic], and then place into tissue culture flasks.</li>
		<li>Change the culture medium every 3 days to 4 days. Ensure that a confluence of 70% to 80% is reached before proceeding further.</li>
		<li>Detach the cells by incubating with trypsin-EDTA for 2-3 min, and passage at a ratio of 1 million cells per T150 flask.</li>
		<li>Expand the cells to P5. After trypsinization, suspend the cells, count them, and then pellet by centrifuging at 300x g for 5 min.</li>
	</ol>
	</li>
	<li>With a 1,000 &#181;L pipette, resuspend the cells at 20 x 106 cells/mL in 15% GelMA solution. 
	NOTE: Turn off the light of the biological safety cabinet.</li>
	<li>Using sterile gloves, press a sterile, dry silicone mold against a Petri dish. Then, put one insert into each hole of the silicone mold with forceps, with the hole side of the insert facing down.</li>
	<li>Using a 200 &#181;L pipette, add the cell suspension to fill up the insert (~50 &#181;L per insert).</li>
	<li>Use a UV flashlight (LED light with a wavelength of 395 nm) to crosslink the top of the gel/insert for 1.5 min. Then, illuminate the other side for 30 s. Crosslinking occurs when the LAP photoinitiator is exposed to UV light. 
	NOTE: The cell suspension may be kept in the incubator during this period or protected from light.</li>
	<li>With sterile forceps, immediately transfer the inserts into 8 mL of growth medium in a non-tissue culture 6-well plate (DMEM supplemented with 10% [v/v] FBS and 1x antibiotic-antimycotic) to allow the cells to recover overnight.</li>
	<li>Differentiate the cells toward four lineages.
	<ol>
		<li>To engineer adipose tissue (AT), transfer the inserts to 8 mL of adipogenic medium (AM; Alpha-MEM, 10% FBS, 0.2 mM indomethacin, 1x insulin-transferrin-selenium (ITS), 0.45 mM 3-isobutyl-1-methylxanthine,&#160;0.1 &#956;M&#160;dexamethasone, and 1x antibiotic-antimycotic) to initiate differentiation. Ideally, four inserts are placed in a single well of a non-tissue culture well plate with 8 mL of adipogenic medium. Culture the cells in the well plates for 28 days, with medium changes every other day.</li>
		<li>To engineer the osteochondral units (OC), place the inserts into dual-flow bioreactor chambers, cap the wells, and infuse the two streams separately at a flow rate of 5 &#181;L/min with 35 mL of osteogenic medium (OM; DMEM, 10% FBS, 1x antibiotic-antimycotic, 0.1&#160;&#956;M dexamethasone, 0.01 M &#946;-glycerophosphate, 100 ng/mL bone morphogenic protein 7 (BMP7), 50 &#181;g/mL ascorbic acid, and 10 nM vitamin D3) and 35 mL of chondrogenic medium (CM; DMEM, 1x antibiotic-antimycotic, 1x&#160;ITS, 0.1&#160;&#956;M&#160;dexamethasone, 40 &#181;g/mL proline, 50 &#181;g/mL ascorbic acid, and 10 ng/mL transforming growth factor &#946;3)10. Maintain cell differentiation for 28 days by performing biweekly medium changes.</li>
		<li>To derive the fibroblasts, differentiate the MSCs in 2D culture over 21 days in a T150 cm2 tissue culture flask using 20 mL of fibrogenic medium (FM; advanced DMEM, 5% FBS, 1x GlutaMAX, 1x antibiotic-antimycotic, and 50 &#181;g/mL ascorbic acid). Change the medium every week. Use 4 mL of trypsin to detach the cells, and encapsulate the 3D gels within the inserts, following the protocol described above, to obtain synovial like-fibrous tissue (SFT). 
		NOTE: The compositions of all the differentiation media can be found in Table 1.</li>
	</ol>
	</li>
</ol>
3. Establishing the miniJoint chip
<ol>
	<li>Autoclave the 3D miniJoint bioreactor chambers, silicone tubing with a 0.062 inch inner diameter and a 0.125 inch outer diameter, and F 1/16 Luer-lock connectors. Connect the silicone tubing to the miniJoint bioreactor barb at one end, and connect the Luer lock at the other end.</li>
	<li>Prepare the AM, OM (removing BMP7), and FMCM mentioned in step 2.11. Additionally, prepare the common shared medium (SM; phenol red-free DMEM, 1x antibiotic-antimycotic,&#160;1x Na-pyruvate, 1x ITS, 40 &#181;g/mL proline, 50 &#181;g/mL ascorbic acid, and 0.5 ng/mL transforming growth factor &#946;3) to be used for the miniJoint culture. Load 35 mL of each medium into the medium reservoirs.</li>
	<li>Use straight forceps to transfer the osteochondral unit from the dual-flow bioreactor to the right well of the miniJoint bioreactor. Transfer the adipose tissue insert and fibrous tissue insert into the left and middle wells, respectively. Cap all the wells with sterilized lids.</li>
	<li>Connect the inlets of the miniJoint chip to the medium reservoirs and the outlets to syringes (Figure 3A-C).</li>
	<li>Mount the syringes onto a syringe pump (Figure 3C), and transfer the pump and chips to an incubator. The medium reservoirs are kept on ice outside the incubator.</li>
	<li>Operate the pump in the withdraw mode, drawing the medium from the medium reservoir into the miniJoint bioreactor chamber. This miniJoint culture process lasts 28 days.</li>
	<li>To model joint inflammation and cartilage degeneration, add interleukin 1&#946; (IL-1&#946;) to the fibrogenic medium stream at 10 ng/mL. Replace the syringes on the third day of IL-1&#946; treatment, and provide fresh IL-1&#946; to the fibrogenic medium. The treatment lasts for 7 days.</li>
	<li>During the drug testing step, after 3 days of IL-1&#946; treatment, administer the drug either in the shared medium, simulating an &#34;intraarticular administration&#34; (Figure 3D) when the drug is used locally in the knee joint, or in all the medium types, simulating a &#34;systemic administration&#34; (Figure 3E) when the drug acts on the knee joint through the circulation.</li>
	<li>Collect individual tissues for analysis after 7 days of IL-1&#946; treatment whether or not the samples underwent drug treatment for the previous 4 days.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64186/64186fig03.jpg" /> 
Figure 3: Assembly of the miniJoint. (A,B) Tissue-specific media are introduced from inlets 1-3 (I1-3) and moved out from outlets 1-3 (O1-3). The shared medium is perfused from I4 to O4. (C) The full setup of the miniJoint culture. Drugs (green sun like shapes) can be either introduced into (D) the shared medium only or (E) all the mediums to respectively simulate &#34;intraarticular administration&#34; or &#34;systemic administration&#34;. <a href="https://www.jove.com/files/ftp_upload/64186/64186fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Individual tissue collection
<ol>
	<li>Use sterile curved forceps to remove the inserts.</li>
	<li>Push a biopsy punch through the center of the insert to remove the gel, and place the gel in PBS.</li>
	<li>Cut the osteochondral gels in half when assessing the gene expression. 
	NOTE: Since the osteochondral gel consists of two tissue types, it is important to separate the osteogenic and chondrogenic cells.</li>
	<li>Collect the conditioned media and tissues for various experiments.
	<ol>
		<li>Collect around 1.5 mL from each medium source.</li>
		<li>Flash-freeze the conditioned media in liquid nitrogen after centrifuging at 14,000 x g for 10 min and discarding the sediment.</li>
		<li>For histological staining and immunostaining, first fix the OC and SFT samples in 10% buffered formalin, dehydrate them in ethanol of ascending concentrations, clear them in xylene, embed them in paraffin, and finally section them at a thickness of 6 &#181;m.</li>
		<li>For the AT microtissues, fix the samples in 10% buffered formalin, and directly stain them with Oil Red O solution or BODIPY.</li>
	</ol>
	</li>
</ol>

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The authors declare no competing interests.

In this article, we present a protocol for creating a knee joint-on-a-chip system, in which bone, cartilage, adipose tissue, and synovium-like tissues are formed from MSCs and co-cultured within a customized bioreactor. This multi-component, human cell-derived system with plug-and-play features represents a new tool for studying the pathogenesis of joint diseases and developing drugs. 
Given that different tissues favor specific culture media, it is critical to provide the respective medium fo...

Joint diseases like osteoarthritis (OA) are highly prevalent and debilitating and represent a leading cause of disability worldwide1. It is estimated that in the US alone, OA affects 27 million patients and occurs in 12.1% of adults aged 60 and above2. Unfortunately, most drugs currently used to manage joint diseases are palliative, and no effective disease-modifying OA drugs (DMOADs) are available3. This unmet medical need primarily stems from the absence of an effective model for studying the disease mechanisms and developing potential DMOADs. The conventional two-dimensional (2D) cell culture does not reflect the 3D nature of joint tissues, and the culture of tissue explants is often hindered by significant cell death and usually fails to replicate the dynamic tissue interconnections4. In addition, genetic and anatomical differences significantly reduce the physiological relevance of animal models4.
Organs-on-chips (OoCs), or microphysiological systems, are a promising research field at the interface of engineering, biology, and medicine. These in vitro platforms are minimal functional units that replicate defined healthy or pathological features of their in vivo counterparts5. Furthermore, these miniaturized systems can host diverse cells and matrices and simulate the biophysical and biochemical interactions between different tissues. Therefore, a microphysiological system that can faithfully recapitulate the native synovial joint promises to offer an effective platform for modeling joint diseases and developing potential DMOADs.
Human mesenchymal stem cells (MSCs) can be isolated from many tissues throughout the body and differentiated into osteogenic, chondrogenic, and adipogenic lineages6. MSCs have been successfully used to engineer various tissues, including bone, cartilage, and adipose tissue6, thus meaning they represent a promising cell source for engineering the tissue components of the knee joint. We recently developed a miniature joint-mimicking microphysiological system, named miniJoint, that comprises MSC-derived bone, cartilage, fibrous, and adipose tissues7. In particular, the novel design enables tissue crosstalk by microfluidic flow or permeation (Figure 1). Herein, we present the protocols for the fabrication of the chip components, the engineering of the tissue components, the culture of the engineered tissues in the chip, and the collection of tissues for downstream analyses.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64186/64186fig01.jpg" /> 
Figure 1: Schematic of the miniJoint chip showing the arrangement of the different tissue components and medium flows. OC = osteochondral tissue. <a href="https://www.jove.com/files/ftp_upload/64186/64186fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-isobutyl-1-methylxanthine</td>
			<td>Sigma -Aldrich</td>
			<td>I17018-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well non-tissue culture plate</td>
			<td>Corning Falcon&#174; Plates</td>
			<td>351146</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well non-tissue culture plate</td>
			<td>Corning Falcon&#174; Plates</td>
			<td>351147</td>
			<td></td>
		</tr>
		<tr>
			<td>30 mL syringes</td>
			<td>BD Syringe Luer Lock Cascade Health</td>
			<td>302832</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian blue stain</td>
			<td>EK Industries</td>
			<td>1198</td>
			<td>1% w/v, pH 1.0</td>
		</tr>
		<tr>
			<td>Advanced DMEM</td>
			<td>Gibco</td>
			<td>12491-015</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;MEM</td>
			<td>Gibco</td>
			<td>12571-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy punch</td>
			<td>Integra Miltex</td>
			<td>12-460-407</td>
			<td></td>
		</tr>
		<tr>
			<td>BODIPY&#174; fluorophore</td>
			<td>Molecular Probes</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bone morphogenic protein 7 (BMP7)</td>
			<td>Peprotech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>Fisher Brand</td>
			<td>16100110</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11995-065</td>
			<td>Dulbecco&#8217;s Modified Eagle Medium</td>
		</tr>
		<tr>
			<td>Dexmethasome</td>
			<td>Sigma -Aldrich</td>
			<td>02-05-2002</td>
			<td></td>
		</tr>
		<tr>
			<td>E-Shell 450 photopolymer in</td>
			<td>EnvisionTec</td>
			<td>RES-01-4022</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gemini-Bio Products</td>
			<td>900-208</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>3505-061</td>
			<td></td>
		</tr>
		<tr>
			<td>gelatin from bovine skin</td>
			<td>Hyclone</td>
			<td>1003372809</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution</td>
			<td>Sigma -Aldrich</td>
			<td>SH30588.02</td>
			<td></td>
		</tr>
		<tr>
			<td>indomethacin</td>
			<td>Sigma -Aldrich</td>
			<td>I7378-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium-Ethanolamine (ITS)</td>
			<td>Gibco</td>
			<td>51500-056</td>
			<td></td>
		</tr>
		<tr>
			<td>interleukin 1&#946;</td>
			<td>Peprotech</td>
			<td>200-01B</td>
			<td></td>
		</tr>
		<tr>
			<td>Leur-loc connectors</td>
			<td>Cole-Parmer Instruments</td>
			<td>45508-50</td>
			<td></td>
		</tr>
		<tr>
			<td>L-proline</td>
			<td>Sigma -Aldrich</td>
			<td>115388-93-7</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Sigma -Aldrich</td>
			<td>1003129352</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium bags</td>
			<td>KiYATEC</td>
			<td>FC045</td>
			<td></td>
		</tr>
		<tr>
			<td>Methacrylic Anhydride</td>
			<td>Sigma -Aldrich</td>
			<td>102378580</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered Saline</td>
			<td>Corning</td>
			<td>21-040-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Pointed forceps</td>
			<td>Fisher Brand</td>
			<td>12000122</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon mold</td>
			<td>McMaster-Carr</td>
			<td>RC00114P</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon o-rings</td>
			<td>McMaster-Carr</td>
			<td>ZMCCs1X5</td>
			<td>1mm x 5mm</td>
		</tr>
		<tr>
			<td>SolidWorks</td>
			<td>Dassault Syst&#232;mes SE, V&#233;lizy-Villacoublay, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Blades</td>
			<td>Integra Miltex</td>
			<td>4-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Lagato210P, KD Scientific</td>
			<td>Z569631</td>
			<td>10 syringe racks</td>
		</tr>
		<tr>
			<td>T-182 tissue culture flasks</td>
			<td>Fisher Brand</td>
			<td>FB012939</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 150 mm</td>
			<td>Fisher Brand</td>
			<td>FB012925</td>
			<td></td>
		</tr>
		<tr>
			<td>Transforming Growth Factor Beta (TGF-&#946;3)</td>
			<td>Peprotech</td>
			<td>100-36E</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Flashlight</td>
			<td>KBS</td>
			<td>KB70109</td>
			<td>395 nm</td>
		</tr>
		<tr>
			<td>Vida Desktop 3D Printer</td>
			<td>EnvisionTec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vitamin D3</td>
			<td>Sigma -Aldrich</td>
			<td>32222-06-3</td>
			<td>1,25-dihydroxyvitamin D3</td>
		</tr>
	</tbody>
</table>

creation of a knee joint-on-a-chip for modeling joint diseases and testing drugs

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target joint disorders are mostly palliative. The unmet need for effective disease-modifying OA drugs (DMOADs) has been primarily caused by the absence of appropriate models for studying the disease mechanisms and testing potential DMOADs. Herein, we describe the establishment of a miniature synovial joint-mimicking microphysiological system (miniJoint) comprising adipose, fibrous, and osteochondral tissue components derived from human mesenchymal stem cells (MSCs). To obtain the three-dimensional (3D) microtissues, MSCs were encapsulated in photocrosslinkable methacrylated gelatin before or following differentiation. The cell-laden tissue constructs were then integrated into a 3D-printed bioreactor, forming the miniJoint. Separate flows of osteogenic, fibrogenic, and adipogenic media were introduced to maintain the respective tissue phenotypes. A commonly shared stream was perfused through the cartilage, synovial, and adipose tissues to enable tissue crosstalk. This flow pattern allows the induction of perturbations in one or more of the tissue components for mechanistic studies. Furthermore, potential DMOADs can be tested via either &#34;systemic administration&#34; through all the medium streams or &#34;intraarticular administration&#34; by adding the drugs to only the shared &#34;synovial fluid&#34;-simulating flow. Thus, the miniJoint can serve as a versatile in vitro platform for efficiently studying disease mechanisms and testing drugs in personalized medicine.

All the tissues of the miniJoint were collected to analyze their phenotypes following 28 days of culture in the miniJoint (Figure 4A). This has been detailed in our previous publication7.
Through the use of RT-qPCR, immunostaining, and histological staining, it was confirmed that the tissue-specific phenotypes were well maintained for the individual microtissues (Figure 4). For example, the osseous component of...

Watch this Scientific Journal Video about Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs at JoVE.com

Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs

We provide detailed methods for generating four types of tissues from human mesenchymal stem cells, which are used to recapitulate the cartilage, bone, fat pad, and synovium in the human knee joint. These four tissues are integrated into a customized bioreactor and connected through microfluidics, thus generating a knee joint-on-a-chip.

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target ...

creation-knee-joint-on-chip-for-modeling-joint-diseases-testing

Research

JoVE Journal

Bioengineering

3.6K Views.  University of Pittsburgh School of Medicine. We provide detailed methods for generating four types of tissues from human mesenchymal stem cells, which are used to recapitulate the cartilage, bone, fat pad, and synovium in the human knee joint. These four tissues are integrated into a customized bioreactor and connected through microfluidics, thus generating a knee joint-on-a-chip. 

Video: Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations

Patient-specific simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered by the absence of in vivo imaging technology for clinically acquiring myocardial fiber orientations. The objective of this project was to develop a methodology to estimate cardiac fiber orientations from in vivo images of patient heart geometries. An accurate representation of ventricular geometry and fiber orientations was reconstructed, respectively, from high-resolution ex vivo structural magnetic resonance (MR) and diffusion tensor (DT) MR images of a normal human heart, referred to as the atlas. Ventricular geometry of a patient heart was extracted, via semiautomatic segmentation, from an in vivo computed tomography (CT) image. Using image transformation algorithms, the atlas ventricular geometry was deformed to match that of the patient. Finally, the deformation field was applied to the atlas fiber orientations to obtain an estimate of patient fiber orientations. The accuracy of the fiber estimates was assessed using six normal and three failing canine hearts. The mean absolute difference between inclination angles of acquired and estimated fiber orientations was 15.4 &deg;. Computational simulations of ventricular activation maps and pseudo-ECGs in sinus rhythm and ventricular tachycardia indicated that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.The new insights obtained from the project will pave the way for the development of patient-specific models of the heart that can aid physicians in personalized diagnosis and decisions regarding electrophysiological interventions.

A methodology to estimate ventricular fiber orientations from in vivo images of patient heart geometries for personalized modeling is described. Validation of the methodology performed using normal and failing canine hearts demonstrate that that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.

Patient-specific  simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered  by the absence of in vivo imaging technology for ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis

Destabilization of medial meniscus (DMM) model is an important tool for studying the pathophysiological roles of numerous arthritis associated molecules in the pathogenesis of osteoarthritis (OA) in vivo. However, the detailed, especially the visualized protocol for establishing this complicated model in mice, is not available. Herein we took advantage of wildtype and progranulin (PGRN)-/- mice as examples to introduce a protocol for inducing DMM model in mice, and compared the onset of OA following establishment of this surgically induced model. The operations performed on mice were either sham operation, which just opened joint capsule, or DMM operation, which cut the menisco-tibial ligament and caused destabilization of medial meniscus. Osteoarthritis severity was evaluated using histological assay (e.g. Safranin&#160;O staining), expressions of OA-associated genes, degradation of cartilage extracellular matrix molecules, and osteophyte formation. DMM operation successfully induced OA initiation and progression in both wildtype and PGRN-/- mice, and loss of PGNR growth factor led to a more severe OA phenotype in this surgically induced model.

We describe a protocol for the destabilization of the medial meniscus (DMM) model in mice, an effective tool for osteoarthritis (OA) research. In addition, we have demonstrated that deficiency of progranulincan exaggerate OA development and progression by using this model, indicating that progranulin plays a protective role in the pathogenesis of OA.

Destabilization of medial meniscus (DMM) model is an important tool for studying the pathophysiological roles of numerous arthritis associated molecules ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Oscillation and Reaction Board Techniques for Estimating Inertial Properties of a Below-knee Prosthesis

The purpose of this study was two-fold: 1) demonstrate a technique that can be used to directly estimate the inertial properties of a below-knee prosthesis, and 2) contrast the effects of the proposed technique and that of using intact limb inertial properties on joint kinetic estimates during walking in unilateral, transtibial amputees. An oscillation and reaction board system was validated and shown to be reliable when measuring inertial properties of known geometrical solids. When direct measurements of inertial properties of the prosthesis were used in inverse dynamics modeling of the lower extremity compared with inertial estimates based on an intact shank and foot, joint kinetics at the hip and knee were significantly lower during the swing phase of walking. Differences in joint kinetics during stance, however, were smaller than those observed during swing. Therefore, researchers focusing on the swing phase of walking should consider the impact of prosthesis inertia property estimates on study outcomes. For stance, either one of the two inertial models investigated in our study would likely lead to similar outcomes with an inverse dynamics assessment.

Body segmental inertial properties are required for inverse dynamics modeling. Using an oscillation and reaction board technique, inertial properties of below-knee prostheses were measured. Using direct measures of prosthesis inertia in the inverse dynamics model of the prosthetic leg resulted in lower magnitudes of resultant joint forces and moments.

The purpose of this study was two-fold: 1) demonstrate a technique that can be used to directly estimate the inertial properties of a below-knee ...

Creation of Patient-Specific Silicone Cardiac Models with Applications in Pre-surgical Plans and Hands-on Training

Three dimensional&#160;models can be a valuable tool for surgeons as they develop surgical plans and medical fellows as they learn about complex cases. In particular, 3D models can play an important role in the field of cardiology, where complex congenital heart diseases occur. While many 3D printers can provide anatomically correct and detailed models, existing 3D printing materials fail to replicate myocardial tissue properties and can be extremely costly. This protocol aims to develop a process for the creation of patient-specific models of complex congenital heart defects using a low-cost silicone that more closely matches cardiac muscle properties. With improved model fidelity, actual surgical procedural training could occur in advance of the procedure. Successful creation of cardiac models begins with the segmentation of radiologic images to generate a virtual blood pool (blood that fills the chambers of the heart) and myocardial tissue mold. The blood pool and myocardial mold are 3D printed in acrylonitrile butadiene styrene (ABS), a plastic dissolvable in acetone. The mold is assembled around the blood pool, creating a negative space simulating the myocardium. Silicone with a shore hardness of 2A is poured into the negative space and allowed to cure. The myocardial mold is removed, and the remaining silicone/blood pool model is submerged in acetone. The described process results in a physical model in which all cardiac features, including intra-cardiac defects, are represented with more realistic tissue properties and are more closely approximated than a direct 3D printing approach. The successful surgical correction of a model with a ventricular septal defect (VSD) using a GORE-TEX patch (standard surgical intervention for defect) demonstrates the utility of the method.

Patient-specific models improve surgeon and fellow confidence when developing or learning surgical plans. Three-dimensional (3D) printers generate adequate detail for surgical preparation, but fail to replicate tissue haptic fidelity. A protocol is presented detailing the creation of patient-specific, silicone cardiac models, combining 3D printing precision with simulated silicone tissue.

Three dimensional&#160;models can be a valuable tool for surgeons as they develop surgical plans and medical fellows as they learn about complex cases. In ...