Objective

Recreate Trial 6 epithelial monolayer output.

Materials

Equipment

  • Biosaftey cabinet
  • Nikon SMZ745T Stereoscopic Microscope with Excelis camera
  • 15 cm diameter glass culture dish
  • Curved forceps
  • Osmo1 Osmometer
  • Evos XL Core inverted microscope
  • 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)
  • 1 mL centrifuge tubes (number dependent on how many samples)
  • Corning 25^2 cm culture flasks
  • 60 mm petri dishes
  • 0.22 um filter units
  • 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 Red Sea seawater (FSW)
  • 30 ppt Red Sea artificial seawater (ASW)
  • 70% ethanol
  • 30% 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.

Concentrated Filtered Seawater (Red Sea Salt)

  1. T0 500 mL of MilliPore water, add 40 g of Red Sea Salt.

  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)

Media B: 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) (final concentration = 13.9 mM)
  • 1 mL Pen/Strep (100x)
  • 100 uL Amphotericin B (250 ug/mL)

Protocol

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 cumulative system from recirculating seawater system

  • Use a razor blade to remove the animal from the mussel and tie to a glass slide.

  • Place glass slide and animal in glass mason jars filled with 100 mL of ASW-G solution, parafilm, and puncture holes using sharp forceps. Leave for 21-24 hours a starving them for that time period at 18 C.

2. Bud excsion

  • Remove colonies from ASW-G.

  • Wash colonies and substrate for 30 seconds with 70% ethanol.

  • Immerse colony in 15 cm diameter, glass dish filled with ~20 mL of ASW-PSA.

  • Assess health score. Condition:
    • 0 = dead (no blood flow)
    • 2 = very bad w/ heartbeat
    • 4 = poor (ampullae retracted, high pigmentation, tunic puffy)
    • 6 = fair (part poor)
    • 8 = good (most ampullae extended, normal tunic & pigmentation)
    • 10 = great (all ampullae extended, tunic flat, clear, normal pigmentation, good blood flow in primary bud vasculature)

  • Using a pair of 31G syringe needles, peel open the tunic and remove buds. Every time a bud is removed, place it in a cell strainer that itself is also immersed in the same 10 mL of ASW-G. Use a clicker to keep track of the number of buds you collected.

  • Once you have collected a group of 10 zooids in the cell strainer, rinse all buds for 5 seconds with 30% ethanol followed by a 5 second rinse with ASW-PSA.

  • Using sterile curved forceps, transfer the group of 10 zooids 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 25cm^2 cell culture flask. Make sure to transfer only a small amount of liquid (about a drop) per zooid.

  • Using a glass pasture pipette and vacuum aspirator, remove any excess liquid in the flask by hovering around the zooids.

  • Once flask is dry using a serological pipette take n*2 mL (where n = the number of flasks) and add 2 mL of TCM to each dish. Be sure to not touch the tip of the pipette to the dish 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 100% of the TCM every day.

Data and Obervations

One cumulative system DM20230628_01 dissected at stage C2 at 07-04-2023.

  • System blastogenic stage delayed. Did not mature to stage D.
  • Health condition: 8
  • 2 mL TCM added at 0 days post seeding (dps).
  • pH and osmolarity measured from removed media. If no media replacement no pH or osmolarity was recorded.
Date Flask ID Media monlayer_num/tissue pH mOsmo/kg % media_removed % media_added comments
07-05-2023 DM20230628_01_01 B 0/9 7.60 894 100 100  
07-05-2023 DM20230628_01_02 B 1/10 7.59 893 100 100  
07-05-2023 DM20230628_01_03 B 0/10 7.58 891 100 100  
07-05-2023 DM20230628_01_04 B 1/10 7.58 895 100 100  
07-06-2023 DM20230628_01_01 B 0/9 NA NA 0 0 dropped flask
07-06-2023 DM20230628_01_02 B 1/10 NA NA 0 0  
07-06-2023 DM20230628_01_03 B 0/10 NA NA 0 0  
07-06-2023 DM20230628_01_04 B 1/10 NA NA 0 0  
07-07-2023 DM20230628_01_01 B 0/9 NA NA 0 50  
07-07-2023 DM20230628_01_02 B 1/10 NA NA 0 50  
07-07-2023 DM20230628_01_03 B 0/10 NA NA 0 50  
07-07-2023 DM20230628_01_04 B 1/10 NA NA 0 50