A simple and effective cryopreservation protocol for the industrially important and model organism, Euglena gracilis

Summary Euglena gracilis is a source of high-value natural products. A major factor affecting consistent production of Euglena biomass is strain stability. Cryopreservation is a leading strategy for cell-line storage that helps ensure process reproducibility. We developed a simple cryopreservation protocol for heterotrophically cultured Euglena that enables the recovery of cells after 1 year with a cell viability of ≅80%. This protocol is suitable for labs interested in the long-term preservation of heterotrophic cultures of Euglena and related species.


E. gracilis growth
Timing: 10-12 days This section describes the growth and culturing of E. gracilis cells from stock in preparation for harvesting. Unless otherwise indicated all culturing, harvesting and subsequent sections should be carried out under sterile conditions.
1. Streak out fresh E. gracilis strain Z on glucose supplemented growth media + Agar (1.5%) using a sterile loop in a biosafety cabinet (or equivalent aseptic technique) and grow in the dark for 5-7 days (28 C) ( Figures 1A-1C). 2. Inoculate 200 mL of fresh glucose supplemented growth media ( Figure 1D) with E. gracilis cells (y3.5 3 10 6 cells/mL) from step 1 using a sterile loop and grow heterotrophically in the dark (28 C, 120 rpm) until the glucose concentration is measured below 1 g/L ( Figure 1G; y3 days). This equates to the late log or early stationary growth phase whereby cell count measures y 12 3 10 6 cells/mL ( Figure 1E) or at OD 600 measures y 4.8 ( Figure 1F). a. Growth of cultures can be monitored using a spectrophotometer and/or a cell counter, and glucose can be monitored using a YSI Biochemistry Analyzer 2950 D or equivalent technique ( Figure 1). We recommend monitoring cell morphology using a compound microscope equipped with a camera ( Figure 1H).

Cell harvesting
Timing: 1-2 h This section describes the harvesting of cells for cryopreservation.
3. Transfer 10 mL of the 200 mL culture to a 15 mL conical tube in a biosafety cabinet (or equivalent aseptic technique) using a 10 mL serological pipette.
CRITICAL: 1 mL of sample should be used to assess cell viability. See: post-cryopreservation culturing and cell viability assays step 21.
4. Pellet cells by centrifugation at 500 g for 5 min at 19 C-22 C (Figure 2A). 5. After centrifugation, discard the supernatant and resuspend cells by gently pipetting in fresh, sterile glucose supplemented growth media to a final cell concentration of y 10 3 10 6 cells/mL. 6. Transfer the cell suspension to a sterile conical tube and let rest undisturbed at 19 C-22 C for a 30 min recovery period ( Figure 2B).
CRITICAL: 30 min cell recovery at 19 C-22 C is critical after centrifugation.

Perform cryopreservation treatment according to the CPA treatment below.
Preparation of the cryoprotective agent (CPA)

Timing: 15 min
These steps describe the preparation of the cryoprotectant agent under sterile conditions. 8. Add 2 mL of reagent grade methanol (MeOH) to 8 mL of sterile glucose supplemented growth media to make 10 mL of 20% (v/v) cryoprotective agent (CPA). 9. Filter-sterilize CPA into a 50 mL sterile conical tube using a 0.2 mm filter equipped with a 50 mL syringe ( Figure 3) and keep at 19 C-22 C until use.
CRITICAL: CPA should be prepared fresh on the day of use. If CPA is stored for long periods or exposed to strong light, it can lose efficiency.

Timing: 30 min
These steps describe the treatment of cells with CPA.
10. Transfer 10 mL of E. gracilis cells (y 10 3 10 6 cells/mL) from step 6 into a universal glass vial and add 10 mL of CPA to make a final 20 mL solution containing 10% MeOH (v/v) (Figure 4). 11. Seal vial(s) with parafilm and mix by gently inverting twice.
Note: You can customize the volume at step 10 if the ratio remains 1-CPA:1-cells (v/v). We make extra CPA in case of spillage or when additional aliquots are desired.

Timing: 30 min
This section describes the aliquoting of CPA treated cells.
12. Aliquot 0.5 mL of CPA treated E. gracilis cells into sterile, 2.0 mL plastic screw cap vials.
a. Repeat for a total of 18 vials -the capacity of the Mr. Frosty cooling container. 13. Seal cryo-vials with parafilm and incubate for 15 min at 19 C-22 C ( Figure 5).

Timing: 2 h
This section describes the transfer of CPA treated cells, the preparation protocol for use of the Mr. Frosty and long-term storage of Euglena cells.  Note: For cooling and cryopreservation of cells it is recommended to use the Mr. Frosty cooling device or a similar device like the Cool Cellä which facilitate gradual cooling of cells. We tested an alternative cooling protocol by placing CPA treated cells directly at À80 C followed by LN 2 but cells did not survive.
14. Cooling Phase a. After incubation (step 13), immediately transfer cryo-vials containing CPA treated E. gracilis cells to the Mr. Frosty passive freezing system.

CRITICAL:
Step 14 should only proceed after the Mr. Frosty unit has been pre-treated with 250 mL of isopropanol and undergone overnight equilibration at 4 C. See: Before You Begin, step 3.
b. Place the Mr. Frosty unit at -80 C and allow cells to rest for 1.5 h. i. The cooling rate during this step equates to À1 C/min, and the temperature of the vial contents after 1.5 h is below À50 C ( Figure 6).
15. Cryopreservation phase a. Take the pre-chilled cryopreservation vials containing the pre-chilled E. gracilis cells and quickly transfer them from the Mr. Frosty cooling unit into the pre-chilled (À196 C) cryobox and cryo-rack for long term storage in LN 2 (Figure 7). b. Monitor LN 2 levels bi-weekly to ensure samples remain submerged in LN 2 .
Pause point: Store cryopreserved cells for the desired time ensuring LN 2 levels are maintained. 16. After the cryopreservation period (i.e., 1 year), remove the cryo-rack from LN 2 and transfer the cryo-vials directly to a pre-warmed water bath (35 C, 1-2 min).
CRITICAL: Immerse cryovials to cover vial contents. This step should be done carefully to avoid agitation, and vial contents should be left immersed until cells are completely thawed (1-2 min) ( Figure 8). The optimal temperature for thawing is 35 C (check with an analog thermometer).
17. After thawing, immediately remove cryo-vials from the water bath and sterilize the outer surface with 70% (v/v) ethanol. Rapidly transfer vials to a class II biosafety cabinet. 18. To increase osmolarity and the total volume to 1.0 mL, carefully add 0.5 mL of fresh glucose supplemented growth media to the cell suspension and mix by gently pipetting. 19. Immediately pellet cells by gentle centrifugation (400 g for 3 min at 19 C-22 C) and discard the supernatant. Resuspend the pellet in 1 mL of fresh glucose supplemented growth media by gently pipetting, and incubate for 10 min at 19 C-22 C. a. Repeat step 19 once to remove remaining CPA.

Note:
We have successfully recovered cells after 1 week, 3 months, 6 months and 1 year of cryopreservation.

Post-cryopreservation culturing and cell viability assays
Timing: 8-10 days This section outlines the cultivation and assessment of cell viability for cells that have been reanimated following cryopreservation and storage. As in previous sections steps should be carried out under sterile conditions.

OPEN ACCESS
under the same conditions but with a modified rotation speed, which should be changed to 120 rpm (Figure 9). i. Monitor cell density (cells/mL) and optical density (OD 600 ) during post-cryopreservation culturing. Make morphological observations using a microscope to determine the health and viability of the culture (Figure 1).

Cell Viability Assays
Note: To evaluate cryopreservation success, if resources permit, we recommend testing the success of the cryopreservation protocol at different time intervals (i.e., bi-weekly, monthly, quarterly, yearly) using the indicated cell viability assay.
a. The viability of E. gracilis cells should be assessed using Trypan Blue (TB) and microscopy. This is done three times: (1) Before cryopreservation (step 3 of cell harvesting), (2) immediately after thawing (step 20a) and (3) immediately after the post-cryopreservation culturing period (step 20b). i. TB stains dead E. gracilis cells, which acquire a dark blue appearance. Living (viable) cells maintain their natural color. The following outlines the TB staining procedure (Figure 10).
Note: When working with Trypan Blue follow all safety protocols as indicated by the manufacturer, wear appropriate PPE and discard waste in appropriate vessels.
b. Add 1 mL of E. gracilis cell culture to a 2 mL tube and centrifuge at 2400 g for 5 min at 19 C-22 C and discard the supernatant. c. Add 1 mL of 0.4% TB solution to the cell pellet, mix by gently pipetting, and incubate at 19 C-22 C for 10 min. d. After incubation, centrifuge the sample at 2400 g for 5 min at 19 C-22 C, remove 900 mL of supernatant and replace with 900 mL of distilled water. e. Mix sample gently by pipetting and centrifuge at 2400 g for 5 min at 19 C-22 C to remove supernatant. i. Resuspend cells in 1 mL of distilled water and repeat the washing step three times. f. A minimum of 50 cells should be counted under the microscope (or microscope photo) to calculate viable and dead E. gracilis cells (Figure 1). g. Cell viability (%) is calculated using the following formula: (number of living cells/ number of total cells) 3 100%. To avoid underestimating cell viability due to the presence of dead cells

EXPECTED OUTCOMES
The developed cryopreservation protocol is a simple and straightforward protocol for the long-term maintenance, storage, and recovery of heterotrophically grown E. gracilis strain Z. This protocol allows for long-term genetic stability and a storage strategy for companies and academic labs interested in E. gracilis. Additionally, it allows for easy strain recovery, transfer between locations and provides a useful starting point for developing subsequent methods for the maintenance and storage of other Euglena strains and species.

LIMITATIONS
This protocol is dependent on the health of the starting cell culture, and adherence to the steps outlined above. This method has been optimized for heterotrophically cultured Euglena gracilis strain Z but serves as a starting point for Euglena grown using alternative carbon sources, and other Euglena strains and species.

TROUBLESHOOTING
Problem 1 Growth kinetics are slowed or there is a reduced culture growth rate (step 1).
Potential solution E. gracilis can grow over a wide temperature range (22 C-30 C) but we recommend growing E. gracilis cells at the experimentally optimized growth temperature (28 C). If slower growth rates are observed, it is recommended to check that all growing conditions are consistent with our suggested parameters. Sub-optimal growth can also be a consequence of an unhealthy culture, and therefore its health should be assessed before proceeding with cryopreservation. Healthy E. gracilis cells in liquid culture are elongated and actively moving. If they are rounded or non-motile, cultures should not be used for cryopreservation.

OPEN ACCESS
Potential solution CPA is a critical solution and should be prepared before harvesting cells for cryopreservation. Cryopreservation efficiency drops with CPA that has been stored for extended time frames or when it is exposed to light. It is not recommended to store CPA at 4 C or À20 C as this can also reduce efficiency. In addition, CPA must be mixed with growth media (1v:1v). It cannot be used with water. We have tested alternative CPA solutions (e.g., glycerol, DMSO) and concentrations and found that the final concentration of 10% MeOH is optimal for heterotrophically grown E. gracilis.

Potential solution
Unlike common laboratory organisms (i.e., yeast), E. gracilis does not have a cell wall which makes it sensitive to abrupt temperature changes. As such, the temperature and timing parameters described in this protocol must be strictly adhered to. For example, transfer of cells from LN 2 to the water bath should be done quickly, as extended times at RT will impact recovery. We also tested different recovery temperatures (i.e., 28 C, 35 C and 37 C) and intervals (1, 2, and 3 min) but 35 C and 1-2 min was optimal. If viability remains poor, one must ensure that cells are healthy before harvesting, and that cells are not damaged during resuspension. Again, we recommend using the cell viability assay at the indicated steps before cryopreservation to assist with the evaluation of cell health.

Potential solution
All transferring steps must be carried out aseptically in a class II biosafety cabinet. While a biosafety cabinet is recommended, a laminar flow-hood and/or flame can be used to maintain dead airspace. All equipment, tubes etc. should be sterile prior to use and good aseptic technique should be used throughout.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Scott Farrow (scott.c.farrow@gmail.com).

Materials availability
This study did not generate new unique reagents.

Data and code availability
The published article includes all steps and indications for successful cryopreservation. Relevant cell count, viability information, and accompanying images are provided as a guide.