SOP In vivo cell implantation Starr Lab
PI: Timothy K. Starr
Lab Location: 12-175 Moos Towers
Original issue date: 10/10/12
Revision dates: 3/4/14,
Prepared by: Timothy K. Starr
Approved by: Timothy K. Starr
Table of Contents
SOP In vivo cell implantation Starr Lab ..................................................................................................... 1
Table of Contents ........................................................................................................................................ 1
Overview: .................................................................................................................................................... 1
General Safety Procedures: ......................................................................................................................... 2
Preparing to work in BSC: ...................................................................................................................... 2
Working in BSC ...................................................................................................................................... 3
Cleaning up after working in BSC .......................................................................................................... 3
Tips to prevent contamination ................................................................................................................ 3
Materials ..................................................................................................................................................... 4
All experiments use the following materials .......................................................................................... 4
Experiments requiring anesthesia ........................................................................................................... 4
Procedure .................................................................................................................................................... 5
Preparation steps ..................................................................................................................................... 5
IP and SC Injections................................................................................................................................ 5
IV injections in tail vein .......................................................................................................................... 6
Anesthesia steps (IB, IC and Colonic) .................................................................................................... 6
Surgery - IB injection.............................................................................................................................. 7
Surgery - IC injection.............................................................................................................................. 8
Colonic injection ..................................................................................................................................... 8
Post Surgery ............................................................................................................................................ 8
Peri-operative Record Keeping ............................................................................................................... 9
Post-Surgical Monitoring Records .......................................................................................................... 9
Hazard Identification and Risk of Exposure to the Hazard ........................................................................ 9
Exposure Controls Specific to Above Risk of Exposure .......................................................................... 10
Waste Generated and Disposal Methods .................................................................................................. 10
Spill and Accident Response Procedures .................................................................................................. 10
Notes ..........................................................................................................Error! Bookmark not defined.
References ................................................................................................................................................. 10
Overview:
Six different injection locations are covered in this procedure. Three locations require
anesthesia, two of these also require surgery, and three locations do not require surgery
or anesthesia:
Anesthesia not required
1) Sub-cutaneous (SC) cell implantation in flank of mouse
2) Intra-venous (IV) cell implantation in tail vein of mouse
3) Intra-peritoneal (IP) cell implantation in peritoneal cavity of mouse
Anesthesia required
4) Colonic cell implantation in the distal colon of mice
Anesthesia and surgery required
5) Intra-bursal (IB) cell implantation in bursa surrounding ovaries of mouse
6) Intra-cecal (IC) cell implantation in cecum of mouse
Procedural steps that are only required for a certain injection site will be specified in the
procedures below.
General Safety Procedures:
All in vitro and in vivo procedures should be undertaken in a class 2 biological safety cabinet
(BSC) to ensure sterility. Two BSC hoods will be used, one in MoosT 12-175, and one
in MCB 1-134A.
Wear Personal Protective Equipment (PPE): sterile gloves, eye goggles, lab coat and arm
sleeves.
Gloves must be worn at all times when working with cells and mice. Remove gloves using the
inside-out technique. Dispose of gloves into biohazard waste container to be
autoclaved. Wash hands immediately after removing gloves and before leaving work
area. Never wear gloves outside of the laboratory, or touch doorknobs, telephones,
personal belongings, etc. with gloved hands.
Transport cells by foot in a sealed, leak-proof primary container within a sealed, leak-proof
secondary container with sufficient absorbent material between the two containers (e.g.
Screw cap tube inside a 15mL conical tube). Attach a biohazard label to the outside
listing: name, contact address, phone #, contents, and emergency contact.
Clean any spilled media containing cells by 30-minute exposure with 10% bleach (1:9 v/v)
solution, followed by spraying with 70% vol/vol ethanol.
Preparing to work in BSC:
If working in MCB 1-134E (animal facilities) post a Biohazard sign on door indicating that
unauthorized personnel may not enter during the experiment.
Remove all unnecessary equipment and supplies from the BSC in order to ensure smooth air
flow. Check that air grilles are clear.
Turn on blower before using to remove particulates in the cabinet. Wait at least five minutes.
Wipe down surface of cabinet interior with disinfectant (paper towels soaked with 10% bleach
followed by spraying with 70% ethanol).
Place supplies and needed equipment in the BSC before beginning work to minimize the
number of arm-movement disruptions across the air barrier of the cabinet. Only items
required for the immediate work should be placed in the BSC.
Place absorbent towels and decontaminating solution (70% ethanol) near the BSC to facilitate
quick clean up of spills.
Wipe the exterior of supplies with a disinfectant (70% ethanol), particularly containers
removed from a water bath. Spray items with a fine mist of 70% ethanol, this will
evaporate quickly and leave no wet mess but still be effective. Segregate items that will
remain clean from the ones that may become contaminated.
Wash hands and arms, wear appropriate protective equipment for the work being done and to
prevent skin flora from contaminating your work.
Adjust stool height so that your neck and face are above the sash opening.
Working in BSC
Delay manipulation of materials for approximately one minute after placing the hands/arms
inside the BSC. Do not rest arms on the front grille. Raising arms slightly will lessen
disruption of air flow.
Work as far back in the cabinet as practical - at least four inches inside the front grill edge.
Move arms slowly and limit arm movement in and out of cabinet.
As a general rule of thumb, keep clean materials at least one foot away from aerosolgenerating activities to minimize the potential for cross-contamination. The work-flow
should be from "clean (left) to contaminated or dirty (right) ". Limit the movement of
"dirty" items over "clean" ones.
Remove media with vacuum and replace with serological pipettes.
Cleaning up after working in BSC
Wipe down the surfaces of all containers and equipment with an appropriate disinfectant (70%
ethanol) and remove from the BSC.
Wipe down the cabinet interior with disinfectant (paper towels soaked with 10% bleach
followed by spraying with 70% ethanol.
Leave blower on for several minutes with no activity so that any airborne contaminants will be
purged from the work area.
Remove gloves and dispose in Biological Hazards bag and wash hands.
Tips to prevent contamination
Clean water baths frequently and/or treat water in bath.
Clean the inside of incubators frequently, particularly the water tray. Use treated di water in the
bottom of the incubator or water tray.
Use HEPA filters on incubator CO2 and air intake lines. Replace regularly.
Lab coat sleeves can introduce contaminants to biological safety cabinets and incubators. Use
coats designated for working in the biological safety cabinet or tissue culture area,
launder frequently. Use disposable sleeve guards if contamination has been a problem.
Never pour media, remove with vacuum and replace with disposable pipettes.
Do not leave flasks of waste media in cabinet, clean after every use.
On a regular basis, decontaminate under the air grilles and wherever parts are removable.
Media is commonly splattered on the front grille allowing fungus to grow undetected on
the under surface of the grille.
Decontaminate the surface of carts or trays used to transfer culture flasks between the
incubator and the biological safety cabinet or microscope.
Keep pipette aids cleaned, especially the nosepiece, and replace filters regularly.
Clean and disinfect vacuum tubing. This should be done by sucking up the disinfectant we
place in the bottom of the waste flask after we have emptied it and reattached it.
Keep the water in the incubator's water jacket full. If water levels in the jacket drop, the ceiling
of the incubator will be cooler causing condensate to form. Water then drops onto
shelves and cell culture containers.
Check port plugs and septums for contamination in incubator interior; they may trap moisture
and harbor fungi.
Materials
All experiments use the following materials
Mice CAUTION All mice must be maintained under SPF barrier conditions
Cells for injection resuspended in sterile phosphate buffered saline
Spray bottle containing 70% (vol/vol) ethanol
Needles (21G, 23G, 27G, 30G) and 1 ml syringes
Mouse harness and 500 ml beaker filled with H2O at 44-48ºC (for IV injections, see Fig. 2)
Scale for weighing mice
Experiments requiring anesthesia
3M Vetbond tissue adhesive or surgical staples
Sterile instruments (scalpel, scissors, needle holders and forceps)
Sterile Petri dishes
Sterile stock solutions of anesthetizing reagents:
ketamine hydrochloride (100 mg/ml)
xylazine hydrochloride (100 mg/ml)
acepromazine (10 mg/ml)
saline solution (0.9% NaCl)
Ketoprofen (brand name ketofen).
Betadine solution (for IC injections)
Dissecting microscope (for IB injections)
Mouse Coloview Endoscope (for Colonic injections)
Sterile pads and gauze
Procedure
Preparation steps
Weigh mice and calculate anesthesia dosing (see below for cocktail amounts).
Using sterile technique, load cells in PBS into 1cc syringes, keep cells and syringes on ice.
Needle size depends on injection site.
SC = 21G
IP, IV and IC = 27G
IB = 30G
Colonic = 8" flexible stainless steel 30G needle, with 45º bevel (Cadence, Inc. USA)
Place all materials in BSC, including mice.
IP and SC Injections
Grasp mouse firmly on scruff of neck and back so mouse is immobile (Fig. 1).
Insert needle into skin on the flank (SC - Fig. 1A) or into peritoneal cavity (IP - Fig 1B), being
careful to avoid nipple. Insert needle with bevel side up and avoid internal organs when
injecting IP by not inserting needle too far.
Gently remove needle and return mouse to cage.
A
B
Figure 1) Proper technique for SC (A) and IP (B) injections
IV injections in tail vein
Place mouse securely in harness
Dip tail in warm water (45ºC) for 30 seconds (Fig. 2A). Wipe dry with sterile gauze
Insert needle into lateral tail vein and gently push plunger (Fig. 2B).
A
B
Figure 2) Proper technique for IV injections. A) Warm tail by dipping in 45ºC water for 30 seconds, then wipe
with sterile gauze. B) Insert needle in lateral tail vein.
Remove needle, remove mouse from harness, and return mouse to cage.
Anesthesia steps (IB, IC and Colonic)
For injections requiring anesthesia (IB, IC and Colonic), prepare anesthesia cocktail. For a 25
g mouse a single dose of 200 µl is prepared by drawing up the following amounts from
the original bottles and combining in a single syringe:
7.5 µl of 100 mg/ml ketamine (= 30 mg/kg)
1.5 µl of 100 mg/ml xylazine (= 6 mg/kg)
1.25 µl of 10 mg/ml Aceprozamine (= 0.5 mg/kg)
189.75 µl of 0.9% saline solution
Use the Anesthetic dosing Xcel spreadsheet to calculate amount needed for entire
experiment. For the secondary anesthesia, if required, omit the xylazine and
aceprozamine and increase the saline to 192.5 µl.
Load a 23G syringe with appropriate volume of 1º anesthetizing reagent.
Firmly hold mouse by scruff of neck pinched between forefinger and thumb, with tail tucked
under little finger.
Insert needle about 10 mm ventrally from rear leg, taking care to avoid nipples and inject 1º
anesthetizing reagent into the intraperitoneal cavity.
Wait until mouse is sufficiently sedated. Mouse should not have any reflex when toe is
pinched. If surgery lasts longer than 20 minutes, make a second injection using the 2º
anesthetizing reagent (ketamine and acepromazine only).
Surgery - IB injection
Lay animal dorsal side up on sterile gauze with head facing away, tail towards you
Shave or wet fur with 70% alcohol at incision point.
Lift wetted skin using forceps and make small incision in the skin (not the peritoneal wall) with
scissors at dorsomedial position and directly above the ovarian fat pad. The fat pad
should be visible beneath surface of peritoneal wall. Fat pad is recognizable by its
white color in contrast to the dark pink tissue surrounding it. Make a second incision in
the peritoneal wall just above the fat pad.
Place a sterile soaked saline gauze pad on the midline adjacent to the incision. Locate ovarian
fat pad and pull it out and rest it on the gauze. Stabilize the ovary by clamping fat pad
with a bulldog clip.
Under a dissecting microscope, position ovary as to allow for insertion of a 30 G needle into
the oviduct tubule bend leading to the bursa. The needle should be inserted close
enough to the infiundibulum to see it under the bursa and the ovary. When needle is
inserted into proper position, it should be visible under the bursa.
Gently push plunger of syringe to inject between the bursa and the ovary while the syringe is
position at the injection site. This works best with two people. One person holds the
needle in place while the second person pushes the plunger. A maximum of 10 – 20 µl
can be injected.
Remove the needle quickly to seal puncture site, but gently enough not to tear the bursa and
tubule. Bursa should appear to be slightly distended with proper injection.
Release fat pad from bulldog clip and gently replace ovaries into peritoneal cavity using
forceps. Gently close body wall by pulling the upper peritoneal lining over the lower
lining. Optional to close incision in peritoneal wall with 2 – 4 sutures using 6-0 silk or
Vicryl sutures.
Close skin with surgical staples or wound clips.
Place animal back in cage with a heat source to avoid hypothermia and speed up recovery.
Surgery - IC injection
Shave lower third of abdomen (can be done before anesthesia)
Lay animal ventral side up on sterile gauze with head facing away, tail towards you
Rub betadine solution on shaved skin 3 times.
Drape abdomen with sterile gauze in a surgical fashion, leaving only shaved skin uncovered.
Make a small incision in the skin 1 cm anterior to the anus. With a forceps, grasp muscular wall
and pull up through the incision. Insert one blade of scissors into hole and use it to push
the intra-abdominal contents away as the blade is inserted in the anterior direction. Use
the scissors to extend the incision an additional 2-3 cm
Identify the cecum and pull up through the incision. Isolate the Cecum by resting on a pre-cut,
sterile gauze. Bath the cecum in warm saline to keep moist.
Inject 50 µl volume of cells into the cecal wall using a 27G syringe. Remove needle. Inspect
injection site to make sure no leakage occurs.
Return cecum to abdominal cavity
Close the abdominal wall using three interrupted stitches using 3-0 or 4-0 sized suture, or use
a running stitch.
Place animal back in cage with a heat source to avoid hypothermia and speed up recovery.
Colonic injection
The Mouse Coloview endoscope (Karl Storz Endoskopy, Tuttlingen, Germany) consists of a
miniature endoscope (1.9 mm outer diameter), a xenon light source, a triple chip
camera, and an air pump to achieve regulated inflation of the mouse bowel. The
procedure is viewed on a color monitor and digitally recorded on tape. This procedure
requires two people, one manipulating the endoscope and one manipulating the syringe
Lay anesthetized mouse on sterile gause.
Insert 8" flexible needle through a Leur lock screwed on the working channel of the endscope
to avoid air leakage.
Insert scope into mouse colon and inflate colon.
Gently push needle to the scope's front. Gently insert the needle into the submucosa with the
open side of the bevel heading up in a flat angle. Inject total volume of 50 µl cell
solution.
Remove needle and remove scope.
Post Surgery
Immediately following surgery mice will be given a subcutaneous injection of Ketoprofen to
reduce pain. We will use a dose of between 2 to 10 mg/kg, starting with 5 mg/kg.
Mice will be monitored daily for wellness, palpable tumors and any procedure-related
complications such as organ failure, thrombosis or ischemia. Incision will be monitored
for swelling, exudate, pain or dehiscence.
Antibiotics will be administered if there are post-procedural infections, according to RAR
procedures.
Ibuprofen will be administered if there are signs of pain. Administration will be 10 ml Children’s
Motrin in 500 ml drinking water and maintained daily until signs of pain are gone.
Remove staples or wound clips 7 or more days post surgery.
Sacrifice mice when moribund, at tumor endpoint, or at 18 months. See original protocol for
detailed description of endpoints and signs of distress.
Peri-operative Record Keeping
For all surgical and anesthetic procedures, a real time procedural record will be kept and
maintained as part of the animal’s records. Records will be maintained for three years to
cover the life of the protocol. Records will include:
* Animal or group identification, protocol number, and the date of the procedure (identical
surgeries on multiple animals will be listed in one record),
* All drugs or fluids administered, including dose, route, time, and the identity of the person
administering the drugs,
* A description of the surgical procedure and identification of the surgeon(s),
* Ongoing findings during monitoring (eg. Heart rate, depth of anesthesia). For surgeries on
more than one animal conducted during the same surgical setup period, findings may
be noted after the end of the surgery.
* Notation of any variations from the normal and expected events during the anesthetic and
recovery periods, including the actions taken and the time performed, the animal’s
response to these actions, and the identity of the person performing these actions,
* Assessment for pain and distress,
* Actions taken to alleviate pain and distress, including non-pharmacologic interventions,
and the response to these actions,
* A notation defining the end of the monitoring period (euthanasia or functional recovery
from the sedation or anesthesia), including the time, date, and the identity of the person
making the notation.
Post-Surgical Monitoring Records
Post operative records will include the time of monitoring, any observed problems, what type of
analgesics/antibiotics are given, if any (including dosages and route), whether the
animals are active, eating, drinking and whether the incision site is healing well or is
infected, animal posture, fur coat texture (indicators of pain), urine and feces production.
These records will be kept for at least 3 days post-surgically for minor surgical
procedures or if the surgery is more invasive, until the post operative period is at an end
(for example, when sutures are removed and surgical wounds are adequately healed).
All records will be readily available to the personnel involved in post surgical monitoring,
the veterinary staff, the IACUC, and federal regulatory officials.
Hazard Identification and Risk of Exposure to the Hazard
The main hazards in this protocol include exposure to sharps and live animals
Exposure Controls Specific to Above Risk of Exposure
Never recap needles, but always place in Sharps disposal units (U Stores # CX40245,
MS07407 or similar), No Cardboard sharps pouches..
Always wear PPE including lab coats, hair net, face shield, gloves, and tyvek sleeves.
Perform all cell work in a BSC.
Use secondary containment when transporting cells or other reagents.
Waste Generated and Disposal Methods
Liquid waste will be collected in a flask or beaker containing bleach 10% (v/v) and will soak for
30 minutes before being sewered.
Solid waste (petri dishes, pipette tips, other disposables) will be disposed of in a Biohazards
bag and incinerated.
Sharps containers will be sealed when ¾ full and placed in designated waste area.
Refer to the Biological Waste Disposal procedures posted on the Tissue Culture room door for
more information
Spill Response Procedures
For spill, splash or aerosol clean up:
Use paper towels to cover then gently and carefully soak them with 1/10 total volume with
appropriate decontaminating agent.
When decontaminating is finished carefully place any and all materials in appropriate
biohazard bin/bag or autoclave bag
Refer to the Biological Decontamination and Spill Clean-up Plan posted on the Tissue culture
room door for more information
Accident Response Procedures
If Incident Results in a Hazard Exposure ( i.e. face or eye splash, cut or puncture with sharps,
contact with non-intact skin):



Encourage needle sticks and cuts to bleed, gently wash with soap and water for 5 minutes; flush
splashes to the nose, mouth, or skin with water; and flush eyes at the nearest eyewash station
with clean water for 15 minutes.
Call 911 or seek medical attention.
o For urgent care employees may go to HealthPartners Occupational and Environmental
Medicine (M/F day time or Urgent Care after hours), or UMMC-Fairview Hospital (24
hrs). You may seek medical attention at the closest available medical facility or your own
healthcare provider.
o Follow-up must be done by HealthPartners Occupational and Environmental Medicine.
Report the incident to your supervisor as soon as possible, fill out the appropriate documentation.
o Employee First Report of Injury
o Supervisor Incident Investigation Report


Send Incident Report Form to the IBC if exposure has occurred during work on an IBC protocol.
Report all biohazard exposures to the Office of Occupational Health and Safety (612-626-5008)
or uohs@umn.edu.
Note: It is important to fill out all of the appropriate documents to be eligible to collect workers
compensation should any complications from the hazardous exposure arise in the
future.
References
For further information view the UMN DEHS website containing Bio Basic Fact Sheets at
http://www.dehs.umn.edu/bio_basicfacts.htm.
For general information on Biosafety, access the Biosafety in Microbiological and Biomedical
Laboratories (BMBL) 5th Edition from the CDC at BMBL
http://www.cdc.gov/biosafety/publications/bmbl5/index.htm
For Material Safety Data Sheets access the Public Health agency of Canada website MSDS
http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/index-eng.php.