Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

Summary

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.

Abstract

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.

Introduction

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 repair^2,3. 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 system^4,5,6. 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's inflammatory response and hypoxic conditions^7,8,9,10. 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.

Protocol

All stem cell and animal procedures were approved by Stanford's Stem Cell Research Oversight committee and by Stanford University's Administrative Panel on Laboratory Animal Care (SCRO-616 and APLAC-31909).

1. Etching of ITO Glass

1. 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.
   1. Remove the excess mask using a blade, leaving only the desired shape of the final ITO design covered on the glass.
2. 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.
   1. Use a hot plate and a thermometer to ensure that the temperature of etching solution is maintained at 70 °C.
3. Once the etching solution reaches 70 °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.
4. Remove the slide from the solution and rinse it with deionized (DI) H₂O.
5. 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 Ω.

2. Preparation of Pyrrole Solution

1. In a 1000 mL glass bottle, dissolve 70 g of sodium dodecylbenzenesulfonate (NaDBS) in 600 mL of DI H₂O.
2. Once the NaDBS is dissolved, add 14 mL of 0.2 M pyrrole and 386 mL of DI H₂O to the solution.
3. Cover the solution with aluminum foil and store at 4 °C.

3. Electroplating of Polypyrrole on ITO glass

1. Warm the pyrrole solution to room temperature until NaDBS is fully redissolved.
   NOTE: This takes about 15 to 20 minutes.
2. Pour 25 mL of the pyrrole solution into a beaker.
3. Connect the etched ITO glass to a multimeter and submerge the slide into the pyrrole solution.
   1. Ensure that the side with 0 Ω resistance is facing towards the platinum mesh reference electrode.
4. 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).
5. Remove the ITO glass from the multimeter and wash electroplated-PPy in DI H₂O to remove residual surfactant.
6. Gently detach the electroplated-PPy plate from the ITO glass using a razor blade.
   1. Store the PPy plate at room temperature.

4. Preparation of Polydimethylsiloxane (PDMS)

1. 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.
2. Remove the bubbles from the molds using a vacuum chamber for 15 - 20 minutes.
3. Place the molds in a 70 °C oven for 3 h.
4. Once cooled, use a flat spatula to remove the PDMS from the molds.

5. Fabrication of the In Vitro Electrical Stimulation Chamber

1. Autoclave the metal plates (WxL, 2.5 cm x 12.5 cm) and flow valves at 120 °C for 1 hour.
2. 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's perimeter.
   1. 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.
3. Layer the materials as following from bottom to top: (1) Metal plate, (2) PDMS (without cutouts), (3) PPy plate (perpendicular to PDMS), (4) PDMS (with cutouts), and (5) cell chamber.
4. Clamp the apparatus together once it is fully aligned.
5. 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.
6. Once the silver paste is cured, reinforce and seal the wire connection with epoxy.
   1. Record the resistance using a multimeter to make sure that the applied field is the same in each chamber.
   2. 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.

6. Plating human Neural Progenitor Cells (hNPCs) on PPy

1. Sterilize the assembled chambers under UV light for 2 hours.
   1. After 1 hour, rotate the assembled chambers 90°.
2. After sterilization, coat the surface of PPy at the bottom of the chamber with 800 µL of a coating substrate and allow the substrate to solidify on the PPy plate in an incubator at 37 °C at 5% CO₂ for 1 h.
3. 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.
4. 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.
5. Collect hNPC pellets using centrifugation at 8000 x g for 5 minutes.
6. Using a hemocytometer, count and resuspend the cells at a volume of 100,000 cells/cm².
7. Plate 100,000 cells into each chamber well (100,000 cells/cm² in 1 mL of media).
8. Return chambers to incubator at 37 °C at 5% CO_2.

7. Electrical Stimulation of hNPCs

1. One day after seeding the cells on the chambers, use a waveform generator to apply electrical stimulation to the cells.
2. Prior to stimulation, remove old media from each chamber well and replenish with 800 µL of fresh media.
3. 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
4. Apply the stimulation to the cells with a square wave signal at ±400 mV with 100 Hz for 1 h.
5. After the stimulation, remove the wires and incubate the chambers for another day in an incubator set at 37 °C with 5% CO₂.
6. Perform subsequent biological analysis including cell viability and quantitative real-time polymerase chain reaction (qRT-PCR) following the manufacturer's protocol.

8. In Vivo PPy Implantation

1. Perform distal middle cerebral artery (dMCA) occlusion stroke models on T cell-deficient adult male nude rats (NIH-RNU 230 ± 30 g) as described previously². Perform implantation one week post-dMCA occlusion.
2. One day prior to surgery, give ampicillin (1 mg/mL) to the rats in their cage water.
3. Anesthetize the rat in an induction chamber using 0.5 - 3% mg/kg of isoflurane administered via inhalation.
4. Confirm anesthetization of the rat by a lack of toe pinch reflex response.
   1. While the animal is under anesthesia, continue to monitor its membrane color, breathing pattern, and rectal temperature every 15 minutes.
5. Once anesthetization is confirmed, apply artificial tears on eyes of rat to prevent dryness.
6. 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.
7. While the rat is under anesthesia, drill a craniectomy above the left cortex. Open the dura.
   1. Remove the dura mater from the brain using a micro-thin surgical needle.
8. 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.
   1. Place the implant primarily on the penumbral cortex medial to the stroke lesion.
9. After the scaffold is placed, place surgicel over the implant to prevent movement with skin closure.
10. 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.
11. Place the rat in a recovery cage as it regains consciousness.
    1. Never leave the animal unattended while it is unconscious.
    2. Do not place the animal with others until it has fully gained consciousness.
12. 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.
    1. Monitor the animals daily until it appears that they are eating, drinking, grooming, and gaining weight; and then weekly after weight gain.
13. 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   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. 

Results

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 efficacy^14,15. The use of a conductive ...

Discussion

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...

Materials

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