Stereotaxic Transplantation of Neural Cell Suspension into a Mouse with Traumatic Brain Injury

Abstract

Source: Furmanski, O., et al. Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells. J. Vis. Exp. (2019)

In this video, human-induced pluripotent stem cell-derived neural cells are stereotaxically injected into the gray matter-white matter border of the deep cortex in a mouse model of traumatic brain injury. The transplanted cells integrate into the tissue, promoting functional recovery and circuit repair in the injured brain.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Stereotaxic transplantation of cell suspension

1. Begin cell transplantation procedure roughly 24 h after craniectomy.
2. Prepare the cell transplantation equipment and surgical supplies
   1. Fill a 1 mL slip-tip syringe with sterile saline for wound irrigation. Attach a 25 G needle to the syringe to control irrigation.
   2. Fill a 1 mL slip-tip syringe with CsA (Cyclosporine A) solution for immunosuppression. Attach a 25 G needle or larger to the CsA syringe. Refill the CsA syringe as needed between surgeries.
   3. Prepare glass needles from 1.0 mm OD (Outer diameter) borosilicate glass capillary pipettes using standard methods.
   4. Use fine tweezers to break the needle tips to approximately a 200 µm diameter. Ensure that the cylindrical shaft of the needle is no longer than 2.5 cm.
3. Calibrate the syringe pump for use with a 10 µL syringe. Enter a flow rate of 0.2 µL/min to deliver a total volume of 2.0 µL.
4. Prepare human induced pluripotent stem cell (hiPSC) suspension.
   1. Perform all cell handling in a cell culture BSL-2 (Biosafety level-2) hood using standard sterile handling techniques.
   2. Prepare cell cultures in advance according to standard conditions defined for the cell type.
      NOTE: Experiments shown in this demonstration used various neural phenotype cells derived from hiPSCs.
   3. Gently dissociate the cells into a single-cell suspension using a cell detachment solution, or other preferred enzymatic or chemical means.
   4. Count cells in suspension, then dilute the suspension to 5 x 10⁴ cells/µL in minimal cell culture medium (e.g., DMEM (Dulbecco's Modified Eagle Medium)) in a 1.7 mL flip top test tube.
   5. Observe the following notes for transplantation:
      1. Maintain the cells in suspension at 37 °C for the duration of procedures.
      2. Load the syringe only immediately prior to performing intraparenchymal injection
         NOTE: If the cell suspension is laid on its side, gravity can cause it to settle or cling to the side of the syringe, leading to irregularities in the number of cells injected.
      3. Allot ~5 x 10⁵ cells (10 µL suspension) per mouse if performing multiple cell transplantation procedures in one day.
5. Perform stereotaxic transplantation surgery
   1. Place the mouse in an anesthesia induction chamber connected to an isoflurane vaporizer with a compressed oxygen source. Induce anesthesia with ~3% isoflurane at ~0.7 L/min oxygen.
   2. Place the mouse in a stereotaxic frame with an attached anesthetic nose cone.
      1. Fix the head in place with ear bars and a bite bar, orienting the head so that the skull frontal bone is horizontal. Maintain anesthesia at 1.5%- 2% isoflurane for the duration of the surgery.
   3. Perform preoperative care and aseptic preparation
      1. Apply hydrating ophthalmic ointment containing antibiotics to the eyes using a cotton swab.
      2. Lavage the incision site with sterile saline to clean the site and to loosen sutures. Gently apply 70% ethanol with a cotton swab to sterilize the incision site.
   4. Remove sutures using fine tweezers and ophthalmic scissors. Irrigate surgery site and craniectomy with abundant sterile saline.
      1. If the cortex displays disqualifying characteristics, such as excessive herniation, discoloration, disrupted vascularization, or hemorrhage, the animal should be excluded.
   5. Load the cell transplant syringe
      1. Move cell suspension from the 37 °C incubator to a cell culture biosafety hood. Gently swirl or tap the tube to ensure a homogeneous cell suspension.
      2. Use a micro pipettor to load ~7.5 µL cell suspension into the Hamilton syringe through the plunger end.
         1. Hold the syringe at a ~120° angle with the plunger end facing down. Insert the plunger, taking care not to introduce an air bubble between the suspension and the plunger tip.
      3. Attach the gasket assembly to the pipette needle, then attach the needle to the syringe.
      4. Push the plunger to move the cell suspension into the pipette needle. If there is resistance against suspension outflow, use fine tweezers to break the needle tip to enlarge the diameter.
   6. Attach the syringe to the stereotaxic syringe pump. Advance the plunger to make sure the syringe pump assembly is working properly.
   7. Move the needle into the coordinates for injection.
      1. Align the needle tip to bregma. Set the X and Y coordinates to 0. Then move the needle tip over the craniectomy to 2.0 mm lateral and -1.0 mm posterior to bregma. Touch the needle tip to the dura mater surface and set the stereotaxic coordinate to Z = 0.
      2. Push the plunger to ensure the cell suspension is flowing adequately before introducing the needle into the brain.
      3. Introduce the needle into the brain to a depth of Z = -1.4 mm. These stereotaxic coordinates place the graft at the gray matter-white matter border of the deep cortex.
   8. Start the syringe pump to infuse cell suspension. Set the lab bench timer to 15 min and start the timer. Use a long working distance microscope to monitor cell suspension outflow.
      1. Irrigate the surgery site with sterile saline during injection to maintain tissue hydration.
      2. At 15 min, slowly withdraw the transplantation needle. Irrigate the surgery site with saline and close the incision with sutures.
   9. Perform postoperative care
      1. Discontinue anesthesia. Deliver CsA by subcutaneous injection into scruff at 10 mg/kg dose. Place the mouse in a clean and pre-warmed postoperative cage.
      2. Provide acetaminophen analgesic in the drinking water at 1.0 mg/mL.
         NOTE: Provide analgesia according to the appropriate Institutional Animal Care and Use Committee (IACUC) standard operating protocol (SOP) and in consideration of experimental outcome variables.
      3. Provide a moistened chow food recovery cage to aid in rehydration and recovery.
6. Continue daily CsA injections at 10 mg/kg throughout the survival duration of the mouse.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 ml syringes	Becton Dickinson (BD)	309659
1.7 ml flip top test tubes	Denville	C2170
10 microliter syringe	Hamilton	7635-01
25G Precision Glide syringe needles	Becton Dickinson (BD)	305122
70% Ethanol			Product of choice; varies by region
Acetaminophen oral suspension	Tylenol (Children's)		Dilute to 1 mg/ml in water
Anesthetic vaporizer	Vetland	0521-11-22
Animal handling cloth			Purchase from department store
Betadine	Purdue Products	NDC-67618-151-32
Compressed oxygen			Product of choice; varies by region
Cyclosporine A	Sigma-Aldrich	30024-100mg
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D8418-500ml
DMEM	Invitrogen (ThermoFisher)	A14430-01
Electrical tape	3M Corporation		Purchase from department store
Fine tweezers	Fine Science Tools	11254-20
Forceps	Fine Science Tools	91106-12
Glass capillary pipettes, 1 mm OD, 0.58 mm ID	World Precision Instruments	1B100F-3
High Speed Rotary Micromotor Kit	Foredom Electric Co.	K.1070 - K.107018
Ideal Micro Drill Burr Set Of 5	Cell Point Scientific	60-1000
Impact One Stereotaxic Impactor for CCI	Leica Biosystems	39463920
Isoflurane	Baxter	NDC-10019-360-60
Lab bench timers	Fisher Scientific	14-649-17
Micropipette puller	MicroData Instruments, Inc.	PMP-102	Any puller will suffice
Microscope cover slips	Fisherbrand	12-545-E
Microscope slide mounting medium			Product of choice
Mirror			Purchase from department store
Needle holder hemostat	Fine Science Tools	12002-12
Ophthalmic ointment	Falcon Pharmaceuticals	NDC-61314-631-36
Ophthalmic spring scissors	Fine Science Tools	15018-10
Plastic box			Purchase from department store
Plastic cylinder			Purchase from department store
QSI motorized syringe pump	Stoelting	53311
Removable needle compression fitting	Hamilton	55750-01
Small rodent stereotaxic frame	Stoelting	51925
Small scissors	Fine Science Tools	14060-09
StemPro Accutase	Invitrogen (ThermoFisher)	A1110501
Sterile alcohol prep pads	Fisherbrand	06-669-62
Sterile cotton swabs/Kendall Q-tips	Tyco Healthcare	540500
Sterile saline	Hospira	NDC-0409-1966-07
Stopwatches (2)	Fisher Scientific	06-662-56
Superfrost Plus Gold microscope slides	Fisherbrand	15-188-48
Sutures - 5.0 silk with curved needle	Oasis	MV-682