This draft is version 0.30.04.19.

Objective

Isolate epithelial tissue from wild Botryllus schlosseri. This means the colonies have not been introduced to recirculating seawater system at UWT and instead are dissected withing the same 5 hour window that they are removed from sampling locations.

Materials

Equipment

  • Biosaftey cabinet
  • Nikon SMZ745T Stereoscopic Microscope with Excelis camera
  • 15 cm diameter glass culture dish
  • Aspirator
  • Sharp forceps
  • Osmo1 Osmometer
  • Evos XL Core inverted microscope
  • Spray bottle
  • Pipette wand
  • Conical holder
  • Wide mouth mason jars (~300 mL)
  • 1000 uL pipette
  • 100 uL pipette

Consumables

  • BH Supplies Insulin Syringes U-100 31G 1ml/cc 5/16” (8mm)
  • 15 mL centrifuge tubes (number dependent on how many samples)
  • Corning 6 well plates
  • 60 mm petri dishes
  • 0.22 um filter units
  • 50 mL, 10 mL, and 5 mL serological pipettes
  • 1000 uL pipette tips
  • 100 um cell strainers
  • Penicillin-Streptomycin (100x stock)
  • Amphotericin-B (250 ug/mL stock)
  • Gentamicin (50 mg/mL stock)
  • 75 ppt filtered seawater (FSW)
  • 30 ppt artificial seawater (ASW)
  • 70% ethanol

Solutions

For all solutions below you will need about 1 L of artificial seawater.

For the culture media itself, you will require 0.6 mL of filtered seawater per 35 mM well you intend to seed.

Filtered Seawater

  1. Mix together the below two solutions:
    • To 500 mL of MilliQ water
    • 54.68 g NaCl
    • 1.648 g KCl
    • 2.884 g CaCl2·2H2O - To 500 mL of MilliQ water
    • 22.28 g MgSO4·7H2O
    • 12.2 g MgCl2·6H2O
  2. Filter solution through 0.22 um filter units (ideally this unit collection vessel can accommodate 1 L).

Artificial Seawater

36.6 grams (g) of Red Sea Salt per liter. Note that since salt was not stored properly I had to up the mass relative to the instructions on the bucket (typically 32.8 g/L).

Artificial Seawater - 0.05% Gentamicin (ASW-G)

This solution is essential for the isolation of tunicate individuals 24 hours prior to dissection.

In the biosaftey cabinet add together the following:

  • 500 mL of artificial seawater (~850 mOsmo/kg)
    • Does not have to be autoclaved or filtered
  • 238.8 uL gentamicin (50 mg/mL stock)

Artificial Seawater - 1% Pen/Strep & 0.01% Amp B (ASW-PSA)

This solution is used as the media in which the tunicate is submerged during dissection.

In the biosafety cabinet add together the following:

  • 494.5 mL filtered seawater (~850 mOsmo/kg)
  • 500 uL Amphotericin B (250ug/mL stock solution)
  • 5 mL of Penicillin-Streptomycin (100x)

Tunicate Culture Medium (TCM) with 1.4% Pen-Strep and 0.1% Amp B

In the biosafety cabinet add together:

  • 45.6 mL of L-15 media
  • 21.7 mL of FSW (to adjust media milli-osmolality from ~300 mOsmo/kg to ~850 mOsmo/kg)
  • 2.4 mL fetal bovine serum (FBS)
  • 1 mL HEPES (20 mMol/L)
  • 1 mL Pen/Strep (100x)
  • 100 uL Amphotericin B (250 ug/mL)

Epithelial Cell Isolation

Be sure to have sufficient stocks of the above three solutions prior to following the below procedures.

1. The day prior to bud isolation

  • Using this staging method, identify individuals with zooids at stage C2, average zooid size of 300 μm, colonies that are relatively large (~200 zooids embedded in tunic), and appear health according to health scoring metric.

  • Remove individuals from recirculating seawater system and place each individual separate glass mason jars filled with 100 mL of ASW-G solution for 24 hours, starving them for that time period. Wild colonies will still be attached to substrate (such as mussel), the substrate and colony are both placed in ASW-G dip. Parafilm the top of the jar and puncture holes using sharp forceps. Place the jar in the reservoir of the recirculating system to maintain soln. at 18 C.

  • Prepare coated plates as needed for the next day.

2. Bud excsion

  • Remove colonies from ASW-G.

  • Wash colonies and substrate for 10 seconds with 70% ethanol.

  • Using a fresh razor blade, carefully remove colony from substrate. Make sure to not puncture or cut the tunic.

  • Rinse the colony again fro an additional 20 seconds with 70% ethanol.

  • Immerse colony in 15 cm diameter, glass dish filled with ~20 mL of ASW-PSA. For each new colony microdissection, rinse the 15 mL glass plate with 70% ethanol in triplicate, followed by triplicate rinse of ASW-PSA prior to refilling plate with more ASW-PSA.

  • Under stereomicroscope on lab bench take picture of colony with Excelis camera.

  • Using a pair of 31G syringe needles, peel open the tunic and remove buds. Buds in earlier stages of development (stage A) are more embedded in the tunic than degrading ones (TO). Be sure to not tear the bud itself, this can be done by gently carving the tunic around the bud and then using a blunt portion of the needle to roll the bud out. Every time a bud is removed, place it in a cell strainer that itself is also immersed in the same 20 mL of ASW-PSA. Use a clicker to keep track of the number of buds you collected.

  • Once you have collected a group of 5 or 10 buds in the cell strainer, rinse all buds for 5 seconds with 70% ethanol followed by a 5 second rinse with ASW-PSA.

  • Using sterile curved forceps, transfer the group of 5 or 10 buds to a labeled, 1.5 mL tube containing 1 mL of TCM.

  • Transfer all tubes containing bud tissue to the biosaftey cabinet in TPS for cell seeding.

3. Seeding tissue and cell maintenance

  • In the biosaftey cabinet, pour out contents of one tube into a 60 mm petri dish. Using a 1000 uL pipette tip aspirate into the solution to get the buds back into suspension and pour quickly.

  • Using the same 1000 uL pipette tip transfer buds one at a time to a fresh 60 mm petri dish. Make sure to transfer only a small amount of liquid (about a drop) per bud.

  • Using the same pipette tip, remove any excess liquid from the culturing dish.

  • While you repeat the above two steps with your other samples, let the wells you previously seeded to dry for about ~5 minutes or until there is no visible liquid left.

  • Once all the wells are dry, using a serological pipette take n*3 mL (where n = the number of dishes) and add 3mL of TCM to each well. Be sure to not touch the tip of the pipette to the wells and to have the lowest flow possible when adding in media to not agitate the tissue and have it unadhere from plate.

  • Incubate cells at 20 C

  • Replace 50% of the TCM every 2-3 days