Name: electrically conductive scaffold to modulate and deliver stem cells
Uploaded: 2018-04-13T13:30:00
Description: Watch this Scientific Journal Video about Electrically Conductive Scaffold to Modulate and Deliver Stem Cells at JoVE.com

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 blad...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="623">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">FGF-Basic</td>
			<td style="width:103px;">Invitrogen</td>
			<td style="width:119px;">CTP0261</td>
			<td style="width:188px;">20 ng/mL for working media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Matrigel</td>
			<td style="width:103px;">Corning</td>
			<td style="width:119px;">cb40234a</td>
			<td style="width:188px;">1:200 dilution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">LIF Protein, Recom. Hum. (10 &#181;g/mL)</td>
			<td style="width:103px;">EMD Millipore</td>
			<td style="width:119px;">LIF1010</td>
			<td style="width:188px;">10 ng/mL for working media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Sylgard 184 silicone 3.9 kg</td>
			<td style="width:103px;">Fisherbrand</td>
			<td style="width:119px;">NC0162601</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Hydrochloric acid</td>
			<td style="width:103px;">Fisherbrand</td>
			<td style="width:119px;">SA56-4</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:213px;">Nitric Acid Concentrate (Certified) ACS, Fisher Chemical</td>
			<td style="width:103px;">Fisherbrand</td>
			<td style="width:119px;">SA95</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">ITO Glass</td>
			<td style="width:103px;">Delta Technologies</td>
			<td style="width:119px;">CG-40IN-0115</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Sodium dodecylbenzenesulfonate</td>
			<td style="width:103px;">Sigma</td>
			<td style="width:119px;">289957-1KG</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:213px;">Pyrrole</td>
			<td style="width:103px;">Sigma</td>
			<td style="width:119px;">131709-500ML</td>
			<td style="width:188px;">Protect pyrrole solution from light and room temperature</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:213px;">8 well glass slide chambers</td>
			<td style="width:103px;">Thermo Sci Nuc&#160;</td>
			<td style="width:119px;">125658</td>
			<td style="width:188px;">Detach the cell chamber and keep it under sterilized conditions</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Flat-Surface Bracket, 3&#34;x1&#34;</td>
			<td style="width:103px;">McMaster-Carr&#160;</td>
			<td style="width:119px;">1030A4</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:213px;">TWO PART SILVER PAINT 14G</td>
			<td style="width:103px;">Electron Microscopy Sciences&#160;</td>
			<td style="width:119px;">1264214</td>
			<td style="width:188px;">Mix two parts (1:1) in plastic plate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">DPBS, 1x, with Ca and Mg, No Phenol Red</td>
			<td style="width:103px;">Genesse&#160;</td>
			<td style="width:119px;">25-508C</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">AB2 ArunA Neural Cell Culture Media Kit</td>
			<td style="width:103px;">Aruna Biomedical&#160;</td>
			<td style="width:119px;">ABNS7013.2</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">hNP1 Human Neural Progenitor Expansion Kit</td>
			<td style="width:103px;">Aruna Biomedical&#160;</td>
			<td style="width:119px;">&#160;hNP7013.1</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:213px;">Noncontact Flow-Adjustment Valve, Nickel-Plated Brass, for 3/32&#34; to 5/8&#34; Tube OD</td>
			<td style="width:103px;">McMaster-Carr&#160;</td>
			<td style="width:119px;">5330K22</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Multimeter</td>
			<td style="width:103px;">Keysight&#160;</td>
			<td style="width:119px;">E3641A</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Wavefoam generator</td>
			<td style="width:103px;">Keysight</td>
			<td style="width:119px;">33210A-10MHz</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Pt meshes</td>
			<td style="width:103px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">298107-425MG</td>
			<td style="width:188px;">Reference electrode with dimensions, 1x1 cm</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:213px;">LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells</td>
			<td style="width:103px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">L3224</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:213px;">BDNF</td>
			<td style="width:103px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">Hs02718934_s1</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">THBS1</td>
			<td style="width:103px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">Hs00962908_m1</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:213px;">GAPDH</td>
			<td style="width:103px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">Hs02758991_g1</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">RNeasy Mini Kit (250)</td>
			<td style="width:103px;">Qiagen</td>
			<td style="width:119px;">74106</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">QIAshredder (250)</td>
			<td style="width:103px;">Qiagen</td>
			<td style="width:119px;">79656</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">RNase-Free DNase Set (50)</td>
			<td style="width:103px;">QIAGEN</td>
			<td style="width:119px;">79254</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">iScript cDNA Synthesis Kit, 100 x 20 &#181;L rxns</td>
			<td style="width:103px;">BIORAD</td>
			<td style="width:119px;">1708891</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:213px;">TaqMan Gene Expression Master Mix</td>
			<td style="width:103px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">4369510</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">7-8 Week Old, male, RNU Rats</td>
			<td style="width:103px;">NCI-Frederick</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">4-0 Ethicon Silk Suture</td>
			<td style="width:103px;">eSutures.com</td>
			<td style="width:119px;">683G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Isoflurane</td>
			<td style="width:103px;">Henry Schein</td>
			<td style="width:119px;">29405</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">V-1 Tabletop Laboratory Animal Anesthesia System</td>
			<td style="width:103px;">VetEquip</td>
			<td style="width:119px;">901806</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">Surgicel Original Absorbable Hemostat</td>
			<td style="width:103px;">Ethicon</td>
			<td style="width:119px;">1952</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Lab Standard Stereotaxic Instrument, Rat</td>
			<td style="width:103px;">Stoelting</td>
			<td style="width:119px;">51600</td>
			<td></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:213px;">Kimberly-Clark Professional&#160;Safeskin Purple Nitrile Sterile Exam Gloves</td>
			<td style="width:103px;">Fisherbrand</td>
			<td style="width:119px;">19-063-130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Sterile Drape</td>
			<td style="width:103px;">Medline</td>
			<td style="width:119px;">DYNJSD1092</td>
			<td></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:213px;">Thermo Scientific&#160;Shandon Stainless-Steel Scalpel Blade Handle, Holds No. 20-25 Blades</td>
			<td style="width:103px;">Fisherbrand</td>
			<td style="width:119px;">53-34</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Walter Stern Scalpel Blade Series 300</td>
			<td style="width:103px;">Fisherbrand</td>
			<td>17-654-456</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:213px;">QuantStudio 6 Flex Real-Time PCR System</td>
			<td style="width:103px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">4484642</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">Frazier Micro Dissecting Hook</td>
			<td style="width:103px;">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 ...

Watch this Scientific Journal Video about Electrically Conductive Scaffold to Modulate and Deliver Stem Cells at JoVE.com

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 ...

The overall goal of this procedure is to create a platform for in vitro electrical stimulation of stem cells on a conductive polymer scaffold, with subsequent in vivo implantation. This method can help answer key questions in stroke research and stem cell biology by providing a means to modulate stem cells. The main advantage is that the stem cells can be optimized in vitro, and subsequently implanted.Although this method can provide an insight into stroke recovery, it can also be applied to other systems as a new cell delivery method. Generally, users new to this technique will struggle due to the precision required for cell chamber assembly, and the technique involved with the craniectomy. In preparation, autoclave the 2.5 by 12.5 centimeter metal plates and the flow valves.Next, using a chamber slide as a template, cut two pieces of PDMS. One piece serves to outline of the chamber's perimeter. On the second piece, cut out two adjacent pieces from the inside of the chamber slide.Both pieces should be rectangular and match the chamber slide. Now, layer the materials. Start with the metal plate, then the uncut PDMS, then the polypyrrole plate, oriented perpendicularly, then the PDMS with cut-outs, and finally, the cell chamber.Align the layers carefully, and clamp the apparatus together. Next, cut two lengths of a metal wire to extend from the apparatus to the outside of the incubator that will contain it. 60 centimeter lengths are usually long enough.Next, use silver paste to attach a wire to each end of the polypyrrole plate protruding from the outside of the chamber. Once the silver paste is cured, reinforce and seal the connections using an epoxy. Now, record the resistance across the assembly using a multimeter.The applied field must be the same in each chamber. Next, plate the cells onto the assembly, and electrically stimulate them as described in the text protocol. Prior to implanting the cells, remove the wires from the assembly.Then, unclamp the cell chambers and separate the PDMS from the conductive scaffold. The scaffold is now ready for implanting. One day prior to the surgery, provide the subject animal ampicillin in its drinking water.On the day of the surgery, anesthetize the rat with isoflurane, and confirm the anesthesia with a toe pinch. Now, apply artificial tears to the rat's eyes to keep them moist. After shaving the skull, drape the animal, and set up for a sterile surgery.Wear protective garments, unpack the sterilized tools, and sterilize the scalp. For the craniectomy, drill a hole above the left cortex down to the dura mater. Make the opening slightly larger than 1 by 3 millimeters, the size of the implant.Then, open the dura and remove the dura mater from the brain using a micro-thin surgical needle. Remove the rig from the TC-culture incubator and disable the rigs. Next, carefully implant the conductive scaffold from the in vitro system onto the rat cortex.Then cover the implant with Surgicel to secure it, or it may move during the skin closure. Now, suture the wound shut using 4-0 silk suture. After completing the sutures, subcutaneously inject the rats with buprenorphine SR.Then, place the rat in a recovery cage until it regains consciousness.The post-operative procedure is detailed in the text protocol. Human neural progenitor cells were exposed to electrical stimulation, and then stained with cell viability assays. The green color is indicative of a living cell.The cells were clearly able to survive the electrical stimulation. Factors released from the neuro-progenitor cells that are important in stroke recovery were evaluated from one microgram of total RNA using quantitative RTPCR. After normalizing the results using the delta delta cycle threshold method, they reveal then the exposure to electrical stimulation up-regulated BDNF and THBS1 expression.After watching this procedure, you should have a good understanding of how to create a cell culture system for electrically-conducting stem cells, and subsequent implantation for those stem cells into a rat model. Once it is mastered, the procedure can be done in 45 minutes if it is performed properly. While you're attempting this procedure, it is important to remember to always monitor the animals for proper breathing pattern and membrane color, to ensure proper anesthesia.Don't forget that working with rats and stem cells can be hazardous, and precautions, such as wearing proper PPE, should always be taken.

Research

JoVE Journal

Bioengineering

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...