Here I both describe the procedure that occurred for epithelial tissue isolation from Botryllus schlosseri for trial 16 and maintain this document as a living log for further updates regarding those cultures.

General Information

Date and Time

August 21, 2023

Preparation of epithelial isolation began on 08/21/23 with the quarantine period. Individual S081C001 was set in approximately 60 mL of Autoclaved Artificial Seawater - Gentamicin (0.05%).

August 22, 2023: 0 days post seeding (dps)

S081C001 was dissected for zooids on 08/22/23 at 11:30 AM until about 1 PM.
Zooids were transferred to an aliqout of tunicate culture media (TCM) in batches of 10 for all 28 zooids.
Zooid tissue was seeded at approximately 3 PM.
S081C001 was dissected for intact ampullae on 08/22/23 at 1 PM until about 2:30 PM.
Ampullae tissue was seed at approximately 3:30 PM.
1mL of TCM was added to both T50 cell culture flasks at about 4 PM.

August 23, 2023: 1 dps

Flasks were removed from biosaftey cabinet at 11:30 AM and observed at the EVOS inverted microscope.
Fungal and bacterial contamination were observed in both flasks. 100% of the media removed. Duplicate rinse with 2 mL of TCM. Replaced media with a final third dose of 2m mL TCM
4 pieces of zooids aspirated accidently, left with 14 pieces of tissue adhered to flask.

Source and Handling of Starting Material

All tunicates when collected from marina are transported to UWT in 500 mL Nalgene polycarbonate jars filled completely with local marina water. Jars are placed in a cooler to maintain temperature for 30 minute drive back to UWT (from City of De Moines Marina).
Upon arrival, tunicates are removed from mussel using a razor blade or kept with substrate and tied to 3’’ x 2’’ x 1.2 mm glass slide using cotton thread. Tunicates are immediately placed in UWT recirculating artificial seawater system (16 C, 27 PPT).
Tunicates are fed every other day (MWF) a mix of commercially available phytoplankton and roti-rich.

Colony Information At Dissection 08/22/23

colony_id	establishment_date	sampling_location	stage	zooid_num	health_score
S081C001	07/26/2023	De Moines Marina (47.44, -122.34)	26	A1-A2	7/10

Cell Isolation Technique

Epithelial cell isolation occurred mechanically with the use of a pair of 28 G insulin needles.

Culture Medium Composition

Tunicate Culture Media:

24.5 mL Basal L-15 Media (formulated and initially filtered on 06/12/23, re-filtered after cracking container on 08/21/23)

5 % FBS
2% Penicillin-Streptomycin (stock = 100 U)

11 mL concentrated, filtered artificial seawater (stock = 75 PPT)
500 uL HEPES buffer (stock = 20 mM/L)
50 uL Amphotericin B (stock = 250 ug/mL)

Description of Culture Vessels and Substrates

Corning 25 cm² Cell Culture Flask, Canted Neck, Tissue Culture Treated, Nonpyrogenic (Cat. No. 430639).
No additional coating with substrate.

Temperature and Environment

08/22/23: Flasks kept overnight in biosaftey cabinet in UW Tacoma, Tacoma Paper and Stationary (TPS), room 232.
1. Temperature: Room Temperature (~20 C)
2. Light: 0
3. Humidity: NA
08/23/23: Flasks moved to primary cell culture incubator in UW Tacoma, Science Building (SCI) incubator.
1. Temperature: 18 C
2. Light: 0
3. Humidity: 91%
08/25/23: UW Tacoma, Science Building (SCI) incubator.
1. Temperature: 18 C
2. Light: 0
3. Humidity: 91%

Tissue Seeding Density

Document the number of cells seeded in each culture vessel. Consider testing different seeding densities to observe their effects.

Flask 1:

Flask 2 (ampullae): 0.8 ampullae / cm²

Observations and Growth Monitoring

Regularly document your observations, including cell morphology, attachment, aggregation, or any changes in appearance. Include photographs if possible.

Viability and Proliferation

Note any signs of cell viability (e.g., cell membrane integrity, absence of debris). Monitor cell proliferation by documenting changes in cell density over time.

pH and Nutrient Levels

If possible, measure and record the pH of the medium during the culture. Monitor nutrient consumption by measuring glucose or other nutrients if relevant.

Media Changes and Additions

08/22/23: Added 1 mL of media at 0 dps
08/23/23: Removed 100% of media and replaced with 2 mL of TCM.
08/25/23: Addition of 1 mL of TCM. No media replacement. Final volume 3 mL.
08/29/23: Removed 100% of media and replaced with 2 mL of TCM.
09/06/23: Removed 100% of media and replaced with 1.3 mL of TCM
09/13/23: Removed 100% of media and replaced with 2 mL of TCM

Summary of Results and Outcomes

Summarize the overall outcome of the experiment, including whether you observed growth, viability, or any other changes.

09/06/23: Terminated zooid flask due to excessive contamination. Microbial movement fast and directional, type of microbial contamination unidentifiable. Ampullae flask retained.

Notes and Anomalies

Document any unexpected events, anomalies, or deviations from your initial plan.

08/23/23 (1 dps): Flask contaminated. Therefore removed all media and did duplicate rinse with TCM prior to 100% replacement. Ampullae cells adherence but not as a monolayer, likely cells that migrated out of the ampullae during seeding.
08/24/23 (2 dps): Ampullae tissue pigment is decreasing. Pigment cells likely dying. No significant change to edges of tissue pieces. Some amount of cells adhered but no monolayers.
08/25/23 (3 dps): Tissue imaging could no longer occur. Issues with saving files on EVOS Xl Core inverted microscope.
08/29/23 (7 dps): Morphology of cells that are adhered has not changed. High quality images not taken due to issues with EVOS microscope.

Next Steps and Modifications

Due to observation of contamination at 1 dps, no modifications will be made to the protocol for Trial 17.
Biosaftey cabinet to be cleaned:
1. 70% ethanol all tools in biosaftey cabinet
2. 70% ethanol all surfaces of biosaftey cabinet
3. Use UV sterilization in biosaftey cabinet for 15 minutes
Re-implement basic aseptic techniques:
1. Thoroughly wash hands and upper arms for at least 20 seconds.
2. Avoid using lab coat until it can be laundered.

References and Resources

Protocol based on:

Citation

Rabinowitz, C., & Rinkevich, B. (2004). In vitro delayed senescence of extirpated buds from zooids of the colonial tunicate Botryllus schlosseri. Journal of Experimental Biology, 207(9), 1523-1532. Google Scholar.

Protocol

Materials

Equipment

Biosaftey cabinet
Nikon SMZ745T Stereoscopic Microscope with Excelis camera
Evos XL Core inverted microscope
Aspirator
Spray bottle
Two 15 cm diameter glass culture dish
Smal shallow dish (size not important)
Curved forceps
Pipette wand
1000 mL waste beaker
1000 uL pipette
100 uL pipette

Consumables

Solution preparation: - 0.22 um filter units - Penicillin-Streptomycin (100x stock) - Amphotericin-B (250 ug/mL stock) - Gentamicin (50 mg/mL stock) - 75 ppt filtered seawater (FSW) - Refractometer

During dissection: - BH Supplies Insulin Syringes U-100 28G 1ml/cc 5/16” (8mm) - 100 um cell strainers - 1.5 mL tubes (number dependent on how many samples) - 30% ethanol - Autoclaved 27 ppt artificial seawater (ASW)

During tissue seeding: - Corning 6 well plates or T25 culture flasks - 60 mm petri dishes - 50 mL, 10 mL, and 5 mL serological pipettes - 1000 uL pipette tips - 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

Option A: 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

Option B: - Formulate 500 mL of concentrated salt water (75 ppt) solution from Red Sea Salt. - Verify salinity using refractometer prior to filtering.

Filter solution through 0.22 um filter unit.

Artificial Seawater

36.6 grams (g) of Red Sea Salt per liter to make solution at ~30 ppt.

Artificial Seawater - 0.05% Gentamicin (ASW-G)

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

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

Select an individual (one glass slide) with at least 30 zooids completely adhered to the slide. Health score must be 7 and above. Stage does not matter but document the day of dissection.
Remove individuals from recirculating seawater system
Use a razor blade to clean the glass slide
Spray animal over sink with 70 % ethanol for 5 seconds or until glass slide is completely coated with ethanol
Rinse animal with autocalved artificial seawater until no ethanol is observable.
Place animal in 15 cm glass petri dish containing 60 mL of ASW-G.
Parafilm and perferoate petri dish
Leave overnight for starvation on lab bench at room temperature.
Prepare coated plates if needed for the next day.

2. Tissue Dissection

Remove colony from ASW-G.
Wash colony and substrate for 10 seconds with 30% ethanol.
Rinse colony with autoclaved artificial seawater
Immerse colony in 15 cm diameter, glass dish filled with ~60 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 entire colony (all zooids to be dissected with Excelis camera.
Record blastogenic stage, zooid number, and health score.
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 cell strainer that itself is also immersed in the same 20 mL of ASW-G. 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 30% 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, use a 1000 uL pipette tip aspirate into the solution to get the buds back into suspension and transfer to plastic 60 mm petri dish.
Using the same 1000 uL pipette tip transfer buds one at a time to a fresh culturing 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 dishes you previously seeded to dry for about ~5 minutes or until there is no visible liquid left.
If there is still liquid in culturing dish, use aspirator to remove any excess to make sure tissue sticks to dish.
Once all the wells are dry, add 2 mL of TCM to T25 flasks. 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 100% of the TCM every 2-3 days.