We thank Dr. Kati Andreasson (Department of Neurology and Neurological Science, Stanford University) for use of the qRT-PCR machine. The work was supported by National Institutes of Health Grants K08NS098876 (to P.M.G.) and Stanford School of Medicine Dean's Postdoctoral Fellowship (to B.O.).

All stem cell and animal procedures were approved by Stanford&#39;s Stem Cell Research Oversight committee and by Stanford University&#39;s Administrative Panel on Laboratory Animal Care (SCRO-616 and APLAC-31909).
1. Etching of ITO Glass
<ol>
	<li>Prepare the indium tin oxide (ITO)-covered glass slide by placing a masking agent over the conductive side of the glass. 
	NOTE: Most commercial transparent tapes can be used as a masking agent.
	<ol>
		<li>Remove the excess mask using a blade, leaving only the desired shape of the final ITO design covered on the glass.</li>
	</ol>
	</li>
	<li>Combine 5% (v/v) of nitric acid and 45% (v/v) of HCl in a glass beaker inside a fume hood to prepare the etching solution.
	<ol>
		<li>Use a hot plate and a thermometer to ensure that the temperature of etching solution is maintained at 70 &#176;C.</li>
	</ol>
	</li>
	<li>Once the etching solution reaches 70 &#176;C, place the masked-ITO glass slide into the solution for 2 minutes to remove the excess ITO layer. 
	NOTE: Tape masking protects the ITO layer from the exposure to acid solution.</li>
	<li>Remove the slide from the solution and rinse it with deionized (DI) H2O.</li>
	<li>Carefully remove the mask and measure resistance using a multimeter to ensure that the remaining ITO is intact. Readouts of the desired etched area should be approximately 0 &#937;.</li>
</ol>
2. Preparation of Pyrrole Solution
<ol>
	<li>In a 1000 mL glass bottle, dissolve 70 g of sodium dodecylbenzenesulfonate (NaDBS) in 600 mL of DI H2O.</li>
	<li>Once the NaDBS is dissolved, add 14 mL of 0.2 M pyrrole and 386 mL of DI H2O to the solution.</li>
	<li>Cover the solution with aluminum foil and store at 4 &#176;C.</li>
</ol>
3. Electroplating of Polypyrrole on ITO glass
<ol>
	<li>Warm the pyrrole solution to room temperature until NaDBS is fully redissolved. 
	NOTE: This takes about 15 to 20 minutes.</li>
	<li>Pour 25 mL of the pyrrole solution into a beaker.</li>
	<li>Connect the etched ITO glass to a multimeter and submerge the slide into the pyrrole solution.
	<ol>
		<li>Ensure that the side with 0 &#937; resistance is facing towards the platinum mesh reference electrode.</li>
	</ol>
	</li>
	<li>Apply an electrical current to initiate electroplating of PPy on the ITO surface using a multimeter (current controlled at 3 mA/cm2 for 4 hours).</li>
	<li>Remove the ITO glass from the multimeter and wash electroplated-PPy in DI H2O to remove residual surfactant.</li>
	<li>Gently detach the electroplated-PPy plate from the ITO glass using a razor blade.
	<ol>
		<li>Store the PPy plate at room temperature.</li>
	</ol>
	</li>
</ol>
4. Preparation of Polydimethylsiloxane (PDMS)
<ol>
	<li>Using a spatula and a weigh dish, mix 18 g of base agent with 2 g of curing agent and pour the mixture into two 10 cm x 8 cm glass molds.</li>
	<li>Remove the bubbles from the molds using a vacuum chamber for 15 - 20 minutes.</li>
	<li>Place the molds in a 70 &#176;C oven for 3 h.</li>
	<li>Once cooled, use a flat spatula to remove the PDMS from the molds.</li>
</ol>
5. Fabrication of the In Vitro Electrical Stimulation Chamber
<ol>
	<li>Autoclave the metal plates (WxL, 2.5 cm x 12.5 cm) and flow valves at 120 &#176;C for 1 hour.</li>
	<li>Using a chamber slide as a template, cut two pieces of PDMS. 
	NOTE: One piece of PDMS serves as the perimeter of the cell chamber with cutouts of two adjoining squares from within the cell chamber. The other PDMS piece is an outline of the chamber&#39;s perimeter.
	<ol>
		<li>Cut out the inside cell chamber so that it is square shaped and matches the shape of the cell chamber. Ensure that the overall shape of both pieces of PDMS is rectangular and matches that of the chamber slide.</li>
	</ol>
	</li>
	<li>Layer the materials as following from bottom to top: (1) Metal plate, (2) PDMS (without cutouts), (3) PPy plate (perpendicular to PDMS),&#160;(4) PDMS (with cutouts), and (5) cell chamber.</li>
	<li>Clamp the apparatus together once it is fully aligned.</li>
	<li>Cut two 60 cm pieces of a metal wire and use silver paste to attach one wire to each end of the PPy plate that protrudes from the outside of the chamber. Ensure that the wires are long enough to extend from the apparatus to the outside of an incubator.</li>
	<li>Once the silver paste is cured, reinforce and seal the wire connection with epoxy.
	<ol>
		<li>Record the resistance using a multimeter to make sure that the applied field is the same in each chamber.</li>
		<li>For implantation, remove the wires; unclamp the cell chambers and PDMS and then them separate from the conductive scaffold. The dimension of the implanted scaffolds is approximately 1 mm x 3 mm x 0.25 mm.</li>
	</ol>
	</li>
</ol>
6. Plating human Neural Progenitor Cells (hNPCs) on PPy
<ol>
	<li>Sterilize the assembled chambers under UV light for 2 hours.
	<ol>
		<li>After 1 hour, rotate the assembled chambers 90&#176;.</li>
	</ol>
	</li>
	<li>After sterilization, coat the surface of PPy at the bottom of the chamber with 800 &#181;L of a coating substrate and allow the substrate to solidify on the PPy plate in an incubator at 37 &#176;C at 5% CO2 for 1 h.</li>
	<li>After 1 hour, remove the supernatant from chambers and gently rinse with 1 mL of DPBS +Ca +Mg per well. 
	NOTE: Avoid washing the surface of PPy forcefully as this will cause the detachment of the coating substrate.</li>
	<li>Using fresh cell media, mechanically dissociate cultured cells for use with the chambers from a tissue culture plate with gentle pipetting. 
	NOTE: Cells should be 90 - 100% confluent upon dissociation.</li>
	<li>Collect hNPC pellets using centrifugation at 8000 x g for 5 minutes.</li>
	<li>Using a hemocytometer, count and resuspend the cells at a volume of 100,000 cells/cm2.</li>
	<li>Plate 100,000 cells into each chamber well (100,000 cells/cm2 in 1 mL of media).</li>
	<li>Return chambers to incubator at 37 &#176;C at 5% CO2.</li>
</ol>
7. Electrical Stimulation of hNPCs
<ol>
	<li>One day after seeding the cells on the chambers, use a waveform generator to apply electrical stimulation to the cells.</li>
	<li>Prior to stimulation, remove old media from each chamber well and replenish with 800 &#181;L of fresh media.</li>
	<li>After the media is changed, place the chambers back into the incubator, extend the wires out of the incubator, and attach them to a waveform generator</li>
	<li>Apply the stimulation to the cells with a square wave signal at &#177;400 mV with 100 Hz for 1 h.</li>
	<li>After the stimulation, remove the wires and incubate the chambers for another day in an incubator set at 37 &#176;C with 5% CO2.</li>
	<li>Perform subsequent biological analysis including cell viability and quantitative real-time polymerase chain reaction (qRT-PCR) following the manufacturer&#39;s protocol.</li>
</ol>
8. In Vivo PPy Implantation
<ol>
	<li>Perform distal middle cerebral artery (dMCA) occlusion stroke models on T cell-deficient adult male nude rats (NIH-RNU 230 &#177; 30 g) as described previously2. Perform implantation one week post-dMCA occlusion.</li>
	<li>One day prior to surgery, give ampicillin (1 mg/mL) to the rats in their cage water.</li>
	<li>Anesthetize the rat in an induction chamber using 0.5 - 3% mg/kg of isoflurane administered via inhalation.</li>
	<li>Confirm anesthetization of the rat by a lack of toe pinch reflex response.
	<ol>
		<li>While the animal is under anesthesia, continue to monitor its membrane color, breathing pattern, and rectal temperature every 15 minutes.</li>
	</ol>
	</li>
	<li>Once anesthetization is confirmed, apply artificial tears on eyes of rat to prevent dryness.</li>
	<li>Ensure that aseptic technique is maintained during this procedure by using sterile gloves, and a sterile surgical drape13. Keep sterile surgical instruments within a sterile field.</li>
	<li>While the rat is under anesthesia, drill a craniectomy above the left cortex. Open the dura.
	<ol>
		<li>Remove the dura mater from the brain using a micro-thin surgical needle.</li>
	</ol>
	</li>
	<li>Implant the conductive scaffolds from in vitro system onto the rat cortex. 
	NOTE: For our experiments, we implanted scaffolds from in vitro day 3 after hNPC plating.
	<ol>
		<li>Place the implant primarily on the penumbral cortex medial to the stroke lesion.</li>
	</ol>
	</li>
	<li>After the scaffold is placed, place surgicel over the implant to prevent movement with skin closure.</li>
	<li>Suture the wound shut and subcutaneously inject the rats with buprenorphine SR at a dosage of 1 mg/kg to manage the pain associated with stereotaxic surgery and dMCA occlusion.</li>
	<li>Place the rat in a recovery cage as it regains consciousness.
	<ol>
		<li>Never leave the animal unattended while it is unconscious.</li>
		<li>Do not place the animal with others until it has fully gained consciousness.</li>
	</ol>
	</li>
	<li>Monitor animals daily for signs of pain including body weight loss, decreased food/water intake, reduced grooming/unkempt coat, decreased/increased spontaneous activity, abnormal posture, dehydration/skin tenting, sunken eyes, hiding, self-mutilation, rapid breathing, open mouthed breathing, twitching, trembling, tremor, and vocalization.
	<ol>
		<li>Monitor the animals daily until it appears that they are eating, drinking, grooming, and gaining weight; and then weekly after weight gain.</li>
	</ol>
	</li>
	<li>Early euthanization via CO2 inhalation and a secondary confirmation of euthanization (as to ensure the animal will not revive due to normal oxygen supply post CO2 inhalation) will occur to&#160; &#160;any animal that appears to be not eating, drinking and gaining weight after the initial surgical procedure; appears in pain; or appears unable to complete the behavioral tests due to a larger than expected motor cortex deficit.&#160;</li>
</ol>

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The authors have no conflicts of interest to declare with this work.

Growing evidence has demonstrated the promise of stem cells as a novel stroke therapy. This promise has resulted in a major effort to advance stem cell therapeutics to the bedside with at least 40 ongoing or completed clinical trials. Stroke pathology offers a unique neurological disorder that lends itself to stem cell therapy because after the acute insult, there is no neurodegenerative process preventing recovery. The exact mechanism of stem cell-enhanced stroke recovery remains unclear. Angiogenesis, synaptogenesis, a...

Stroke is the second leading cause of death in the world and the fifth leading cause of death in the United States. Despite these high death rates, treatments for stroke recovery currently remain a challenge with no viable medical options currently available1. There are currently about 300 clinical trials dealing with ischemic strokes, of which only 40 utilize stem cells. Previous studies have shown that stem cell therapies have a beneficial effect on stroke repair2,3. Paracrine factors such as brain-derived neurotrophic factor (BDNF) and thrombospondin-1 (THBS-1) released from transplanted human neural progenitor cells (hNPCs) have shown improved functional recovery through mechanisms associated with an increase in synapse formation, angiogenesis, dendritic branching and new axonal projections, as well as modulating the immune system4,5,6. However, the optimal delivery methods of the stem cells remain elusive.
Successful stem cell delivery to the brain remains a challenge. Currently, injectable hydrogel and polymeric scaffolding systems have been introduced to deliver stem cells. These delivery methods protect stem cells during transplantation as well as offer protection from the harsh post-stroke environment including the host&#39;s inflammatory response and hypoxic conditions7,8,9,10. However, the most commonly used materials are inert, which limits the use of continuous modulation (i.e., electrical stimulation) of the cells11. Electrical stimulation is a cue that influences differentiation, ion channel density, and neurite outgrowth of stem cells12. As compared to inert polymers, conductive polymers can carry a current allowing for electrical stimulation and manipulation of stem cells2. However, the precise mechanism by which electrical stimulation modulates neurotrophic factor release (i.e., BDNF and THBS-1) is still not fully explored.
In this protocol, we describe the steps to construct a cell culture system consisting of a conductive polymer scaffold, polypyrrole (PPy), that allows for in vitro electrical stimulation. Because of the manner in which the cell culture system is fabricated, subsequent implantation of the stem cell-seeded scaffold onto the peri-infarct cortex is possible. For this system, we electrically precondition stem cells on the scaffold for a short time period prior to implantation. Following electrical stimulation, the conductive polymer scaffold carrying the cells is successfully implanted intracranially using a minimally invasive method.

<table><tbody><tr><td>FGF-Basic</td><td>Invitrogen</td><td>CTP0261</td><td>20 ng/mL for working media</td></tr><tr><td>Matrigel</td><td>Corning</td><td>cb40234a</td><td>1:200 dilution</td></tr><tr><td>LIF Protein, Recom. Hum. (10 &amp;micro;g/mL)</td><td>EMD Millipore</td><td>LIF1010</td><td>10 ng/mL for working media</td></tr><tr><td>Sylgard 184 silicone 3.9 kg</td><td>Fisherbrand</td><td>NC0162601</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Fisherbrand</td><td>SA56-4</td><td></td></tr><tr><td>Nitric Acid Concentrate (Certified) ACS, Fisher Chemical</td><td>Fisherbrand</td><td>SA95</td><td></td></tr><tr><td>ITO Glass</td><td>Delta Technologies</td><td>CG-40IN-0115</td><td></td></tr><tr><td>Sodium dodecylbenzenesulfonate</td><td>Sigma</td><td>289957-1KG</td><td></td></tr><tr><td>Pyrrole</td><td>Sigma</td><td>131709-500ML</td><td>Protect pyrrole solution from light and room temperature</td></tr><tr><td>8 well glass slide chambers</td><td>Thermo Sci Nuc&amp;nbsp;</td><td>125658</td><td>Detach the cell chamber and keep it under sterilized conditions</td></tr><tr><td>Flat-Surface Bracket, 3&quot;x1&quot;</td><td>McMaster-Carr&amp;nbsp;</td><td>1030A4</td><td></td></tr><tr><td>TWO PART SILVER PAINT 14G</td><td>Electron Microscopy Sciences&amp;nbsp;</td><td>1264214</td><td>Mix two parts (1:1) in plastic plate</td></tr><tr><td>DPBS, 1x, with Ca and Mg, No Phenol Red</td><td>Genesse&amp;nbsp;</td><td>25-508C</td><td></td></tr><tr><td>AB2 ArunA Neural Cell Culture Media Kit</td><td>Aruna Biomedical&amp;nbsp;</td><td>ABNS7013.2</td><td></td></tr><tr><td>hNP1 Human Neural Progenitor Expansion Kit</td><td>Aruna Biomedical&amp;nbsp;</td><td>&amp;nbsp;hNP7013.1</td><td></td></tr><tr><td>Noncontact Flow-Adjustment Valve, Nickel-Plated Brass, for 3/32&quot; to 5/8&quot; Tube OD</td><td>McMaster-Carr&amp;nbsp;</td><td>5330K22</td><td></td></tr><tr><td>Multimeter</td><td>Keysight&amp;nbsp;</td><td>E3641A</td><td></td></tr><tr><td>Wavefoam generator</td><td>Keysight</td><td>33210A-10MHz</td><td></td></tr><tr><td>Pt meshes</td><td>Sigma-Aldrich&amp;nbsp;</td><td>298107-425MG</td><td>Reference electrode with dimensions, 1x1 cm</td></tr><tr><td>LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>L3224</td><td></td></tr><tr><td>BDNF</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>Hs02718934_s1</td><td></td></tr><tr><td>THBS1</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>Hs00962908_m1</td><td></td></tr><tr><td>GAPDH</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>Hs02758991_g1</td><td></td></tr><tr><td>RNeasy Mini Kit (250)</td><td>Qiagen</td><td>74106</td><td></td></tr><tr><td>QIAshredder (250)</td><td>Qiagen</td><td>79656</td><td></td></tr><tr><td>RNase-Free DNase Set (50)</td><td>QIAGEN</td><td>79254</td><td></td></tr><tr><td>iScript cDNA Synthesis Kit, 100 x 20 &amp;micro;L rxns</td><td>BIORAD</td><td>1708891</td><td></td></tr><tr><td>TaqMan Gene Expression Master Mix</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>4369510</td><td></td></tr><tr><td>7-8 Week Old, male, RNU Rats</td><td>NCI-Frederick</td><td></td><td></td></tr><tr><td>4-0 Ethicon Silk Suture</td><td>eSutures.com</td><td>683G</td><td></td></tr><tr><td>Isoflurane</td><td>Henry Schein</td><td>29405</td><td></td></tr><tr><td>V-1 Tabletop Laboratory Animal Anesthesia System</td><td>VetEquip</td><td>901806</td><td></td></tr><tr><td>Surgicel Original Absorbable Hemostat</td><td>Ethicon</td><td>1952</td><td></td></tr><tr><td>Lab Standard Stereotaxic Instrument, Rat</td><td>Stoelting</td><td>51600</td><td></td></tr><tr><td>Kimberly-Clark Professional&amp;nbsp;Safeskin Purple Nitrile Sterile Exam Gloves</td><td>Fisherbrand</td><td>19-063-130</td><td></td></tr><tr><td>Sterile Drape</td><td>Medline</td><td>DYNJSD1092</td><td></td></tr><tr><td>Thermo Scientific&amp;nbsp;Shandon Stainless-Steel Scalpel Blade Handle, Holds No. 20-25 Blades</td><td>Fisherbrand</td><td>53-34</td><td></td></tr><tr><td>Walter Stern Scalpel Blade Series 300</td><td>Fisherbrand</td><td>17-654-456</td><td></td></tr><tr><td>QuantStudio 6 Flex Real-Time PCR System</td><td>Thermo Fisher Scientific</td><td>4484642</td><td></td></tr><tr><td>Frazier Micro Dissecting Hook</td><td>Harvard Apparatus</td><td>52-2706</td><td></td></tr></tbody></table>

electrically conductive scaffold to modulate and deliver stem cells

Stem cell therapy has emerged as an exciting stroke therapeutic, but the optimal delivery method remains unclear. While the technique of microinjection has been used for decades to deliver stem cells in stroke models, this technique is limited by the lack of ability to manipulate the stem cells prior to injection. This paper details a method of using an electrically conductive polymer scaffold for stem cell delivery. Electrical stimulation of stem cells using a conductive polymer scaffold alters the stem cell's genes involved in cell survival, inflammatory response, and synaptic remodeling. After electrical preconditioning, the stem cells on the scaffold are transplanted intracranially in a distal middle cerebral artery occlusion rat model. This protocol describes a powerful technique to manipulate stem cells via a conductive polymer scaffold and creates a new tool to further develop stem cell-based therapy.

The schematic shown in Figure 1 represents the overall workflow of the electrical stimulation of hNPCs and potential downstream applications. A current limitation in stem cell therapy is that stem cells are exposed to a harsh post-transplantation environment including inflammation and ischemic conditions. These difficult conditions likely limit their therapeutic efficacy14,15. The use of a conductive ...

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Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

This protocol describes fabrication of a cell culture system to allow seeding of stem cells on a conductive polymer scaffold for in vitro electrical stimulation and subsequent in vivo implantation of the stem cell-seeded scaffold using a minimally invasive technique.

Stem cell therapy has emerged as an exciting stroke therapeutic, but the optimal delivery method remains unclear. While the technique of microinjection ...

electrically-conductive-scaffold-to-modulate-and-deliver-stem-cells

Research

JoVE Journal

Bioengineering

12.8K Views.  Stanford University School of Medicine. Dit protocol beschrijft fabricage van een celcultuur dat zaaien van de cellen van de stam op een geleidend polymeer steiger voor in vitro elektrische stimulatie en latere in vivo innesteling van de cel van de stam-geplaatste steiger, met behulp van een minimaal invasieve techniek.

Video: Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) has been demonstrated to increase MSC osteogenic differentiation in vitro and promote bone healing in clinical settings. Here we describe the construction of an EStim cell culture chamber and its use in treating rat bone-marrow-derived MSC to enhance osteogenic differentiation. We found that treating MSCs with EStim for 7 days results in a significant increase in the osteogenic differentiation, and importantly, this pro-osteogenic effect persists long after (7 days) EStim is discontinued. This approach of pretreating MSCs with EStim to enhance osteogenic differentiation could be used to optimize bone tissue engineering treatment outcomes and, thus, help them to achieve their full therapeutic potential. In addition to this application, this EStim cell culture chamber and protocol can also be used to investigate other EStim-sensitive cell behaviors, such as migration, proliferation, apoptosis, and scaffold attachment.

Here we present a protocol for the construction of a cell culture chamber designed to expose cells to various types of electrical stimulation, and its use in treating mesenchymal stem cells to enhance osteogenic differentiation.

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) ...

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Finite Element Modelling of a Cellular Electric Microenvironment

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the mechanisms of cell response when exposed to electrical fields can therefore guide the optimization of clinical applications. In vitro experiments aim to help uncover those, offering the advantage of wider input and output ranges that can be ethically and effectively assessed. However, the advancements in in vitro experiments are difficult to reproduce directly in clinical settings. Mainly, that is because the ES devices used in vitro differ significantly from the ones suitable for patient use, and the path from the electrodes to the targeted cells is different. Translating the in vitro results into in vivo procedures is therefore not straightforward. We emphasize that the cellular microenvironment's structure and physical properties play a determining role in the actual experimental testing conditions and suggest that measures of charge distribution can be used to bridge the gap between in vitro and in vivo. Considering this, we show how in silico finite element modelling (FEM) can be used to describe the cellular microenvironment and the changes generated by electric field (EF) exposure. We highlight how the EF couples with geometric structure to determine charge distribution. We then show the impact of time dependent inputs on charge movement. Finally, we demonstrate the relevance of our new in silico model methodology using two case studies: (i) in vitro fibrous Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT-PSS) scaffolds and (ii) in vivo collagen in extracellular matrix (ECM).

This paper presents a strategy for building finite element models of fibrous conductive materials exposed to an electric field (EF). The models can be used to estimate the electrical input that cells seeded in such materials receive and assess the impact of changing the scaffold's constituent material properties, structure or orientation.

Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

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