Background

The volvocine green algae are composed of the unicellular genus Chlamydomonas and multicellular genera such as Gonium and Volvox (Additional file 1: Fig. S1). Because these green algae represent a unique model lineage for experimental studies of the evolution of sex and multicellularity [1], multicellular volvocine algae have been investigated in molecular and genomics studies [2,3,4]. Among the volvocine green algae, two independent lineages, Volvocaceae (including Volvox) and Astrephomene (Fig. 1), show similar or convergent evolution of multicellular spheroidal bodies with germ-soma differentiation (Additional file 1: Fig. S1) [5,6,7]. Whole-genome sequencing of A. gubernaculifera provided insight into the molecular-genetic basis of such convergent evolution [8]. Thus, Astrephomene represents a hopeful key organism for studies of multicellularity and germ-soma differentiation.

Fig. 1
figure 1

Asexual life cycle of Astrephomene. A Diagram of asexual life cycle of Astrephomene. Based on Nozaki [9] and Yamashita et al. [10]. Colonies are generally 32- or 64-celled with two or four somatic cells in the posterior pole. Each reproductive cell performs cell divisions to produce a daughter colony. B Mature vegetative colony of A. gubernaculifera strain NIES-4017. C Posterior portion of parental colony of A. gubernaculifera strain NIES-4017 showing embryogenesis of reproductive cells and four undivided somatic cells (arrow)

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The genus Astrephomene was originally described by Pocock [11] based on a single species: A. gubernaculifera. Using culture strains of A. gubernaculifera originating from the USA and Mexico, morphology, sexual isolation within the morphological species, and physiology were studied [12,13,14]. The second species, A. perforata, was described based on clonal cultured materials from Japan [9]. A. perforata differs from A. gubernaculifera in the morphology of the individual sheaths of cells in the spheroid and pyrenoids in the chloroplast [9]. Fifteen strains of the two species of Astrephomene established in these studies were deposited in the Culture Collection of Algae at the University of Texas at Austin (CCA-UTEX) [15]. Nine strains of the two Astrephomene species are available from the MCC-NIES (https://mcc.nies.go.jp/index_en.html [16]) and one strain of A. gubernaculifera is available from the Culture Collection of Algae at Goettingen University (SAG) (https://uni-goettingen.de/en/45175.html [17]). Since these culture strains are maintained by serial inoculations of living cells to new media, high costs are carried in the culture collections. However, cryopreserved culture strains of Astrephomene have not been established.

Although Mori et al. [18] examined cell survival after freezing in liquid nitrogen in six strains of two species of Astrephomene maintained in the MCC-NIES [16] (https://mcc.nies.go.jp/index_en.html) by using dimethyl sulfoxide (DMSO) as a cryoprotectant, none survived freezing. Later, Nakazawa & Nishii [19] demonstrated poor recovery (i.e., recovery of one or two of three replicates) of A. gubernaculifera strain NIES-418 after cryopreservation in liquid nitrogen when N,N-dimethylformamide (DMF) or hydroxyacetone (HA) was used as a cryoprotectant for two-step freezing. However, no other studies of the cryopreservation or establishment of cryopreserved strains of Astrephomene have been performed.

This study was undertaken to determine the optimal conditions for cryopreservation of culture strains of two species of Astrephomene. Optimal conditions for the cryopreservation of the two species were determined using immature and mature colonies from the asexual cycle of the two Astrephomene species (Fig. 1) and two cryoprotectants (DMF and HA). By using these conditions, cryopreserved strains of the two species were established.

Materials and methods

Culture strains

Nine culture strains of two species of Astrephomene maintained at the MCC-NIES [16] were used (Table 1). The cultures were grown in screw-cap tubes (18 × 150 mm) containing 10 mL of Volvox thiamin acetate (VTAC) medium or urea soil Volvox thiamin (USVT) medium [16] at 25 °C, with a 12 h:12 h light:dark schedule under cool-white fluorescent lamps at an intensity of 100–130 μmol m− 2 s− 1. To maintain the cultures, USVT medium was used for A. gubernaculifera strain NIES-853, whereas the other six strains of A. gubernaculifera and two strains of A. perforata were cultured in VTAC medium.

Table 1 List of strains of two species of Astrephomene used in this study
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To prepare cultures of immature colonies (newly released small daughter colonies with reproductive cells approximately 5 μm in diameter) (Fig. 2A, C), 0.2–0.3 mL of 4–5-day-old cultures (approximately 106 cells/mL) were inoculated into 10 mL of USVT medium in a screw-cap tube 4–6 h after the onset of the light period of the 12 h:12 h light:dark cycle. The inoculated cultures were incubated for 48 h at 25 °C with a 12 h:12 h light:dark cycle, as described above. Cultures of mature colonies (large colonies just before daughter colony formation, with reproductive cells approximately 15 μm in diameter) (Fig. 2B, D) were obtained as described above, except that the inoculum was diluted 30–50-fold with USVT medium.

Fig. 2
figure 2

Immature and mature colonies of two species of Astrephomene (Fig. 1A) that were used for cryogenic treatments (Table 2), shown at the same magnification throughout. A Immature colonies of A. gubernaculifera strain NIES-4017. B Mature colonies of A. gubernaculifera strain NIES-4017. C Immature colonies of A. perforata strain NIES-564. D Mature colonies of A. perforata strain NIES-564

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Cryopreservation

The optimal cryopreservation conditions for two species of Astrephomene were assessed using DMF or HA as a cryoprotectant. Nakazawa and Nishii [19] demonstrated partial survival of A. gubernaculifera cells after freezing in liquid nitrogen with 3% DMF and 3% HA. Nakazawa and Nishii [19] studied the cryopreservation of multicellular volvocine algae using 0.25 mL PCR tubes as vials for two-step freezing. However, we recently demonstrated that use of 2 mL cryotubes (Cryo.s, 2 mL, Round Bottom, Starfoot Base; Greiner Bio-One, Kremsmünster, Austria) as vials resulted in a higher survival rate than achieved using 0.20 mL PCR tubes for cryopreservation of the multicellular volvocine alga Gonium pectorale [20]. Thus, we prepared 1.0 mL samples in 2 mL cryotubes for cryopreservation of two species of Astrephomene, and eight cryopreservation conditions were examined for A. gubernaculifera strain NIES-4017 and A. perforata strain NIES-564: immature colonies in 3% DMF, immature colonies in 6% DMF, immature colonies in 3% HA, immature colonies in 6% HA, mature colonies in 3% DMF, mature colonies in 6% DMF, mature colonies in 3% HA, and mature colonies in 6% HA. For cryopreservation, a 48-day-old culture of immature or mature colonies (see above) actively growing in USVT medium (2–4 mL) was mixed with an equal volume of USVT medium containing 6% or 12% DMF (or HA) to prepare a sample with 3% or 6% DMF (or HA), respectively. The cells were exposed to the cryoprotectant at room temperature (20–25 °C) for 15 min. Next, 1.0 mL of the culture sample with DMF (or HA) was transferred to a 2 mL cryotube. The sample cryotube was subjected to two-step cooling in liquid nitrogen [18, 20, 21]. Cell suspensions in tubes were frozen in vapor-phase liquid nitrogen at a rate of − 1 °C/min to − 40 °C using a programmable freezer (Controlled Rate Freezer, KRYO 560-16; Planer, Sunbury-on-Thames, UK). After 15 min of maintenance at − 40 °C, the cell suspensions were cooled rapidly to − 196 °C by immersion in liquid nitrogen, and finally stored at − 190 °C in vapor-phase liquid nitrogen. To assess the viability of cells frozen in liquid nitrogen, the frozen samples in tubes were thawed in a 40 °C or 60 °C water bath while the tube was shaken by hand until the ice crystals almost disappeared (approximately 120 or 75 s, respectively); then, 0.1 mL of the diluted sample was immediately subjected to analysis using the most probable number (MPN) method [18, 20,21,22,23]. For the MPN method, eight wells in each dilution series of a 48-well microplate (CellStar Cell Culture Multiwell Plate with Lid, Greiner Bio-One) were filled with 0.9 mL of USVT medium. Three replicates of eight 1/10th dilutions were performed for each cryotube of sample using a 6-channel pipette (Pipet-Lite Adjustable Spacer LA6-1200XLS; Mettler-Toledo, Greifensee, Switzerland). As a control, three replicates of eight 1/10th dilutions of cultures without cryogenic treatment and cryoprotectant were treated in the same manner. The plates were initially incubated in darkness at 25 °C for 2 days, then transferred to a 12 h:12 h light:dark schedule at 25 °C for 2 weeks [20]. Each well was scored for growth and MPN values (cell numbers) were estimated based on those scores using MPN Calculator 3.1 (https://softdeluxe.com/MPN-Calculator-444229/) [24, 25]. The recovery rate of viable cells (%) was calculated relative to the viable cell count in the unfrozen control using the MPN method. For each of the four types of cryopreservation conditions, recovery rates were measured based on six tubes from two independent experiments (Table 2).

Table 2 Comparison of results of eight types of cryopreservation conditions for two species of Astrephomene based on most probable number (MPN) methods
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In addition, immediately after thawing of the three frozen cryotubes of each sample, 0.5 mL of the melted sample in each cryotube was inoculated into fresh growth medium (10 mL) in a six-well plate (PS with Lid; Greiner Bio-One) (first inoculation); subsequently, 0.5 mL of the first inoculation was transferred to 10 mL of fresh growth medium (second inoculation) in a six-well plate to confirm the recovery of frozen and thawed cells in the same volume of culture medium used in the MCC-NIES.

Results

“MPN survival” after the eight cryogenic treatments differed between the two species of Astrephomene (Table 2). For A. gubernaculifera strain NIES-4017, the highest recovery rate after freezing in liquid nitrogen and thawing was achieved when immature colonies were subjected to 6% DMF during two-step freezing (11 ± 13% MPN survival, Table 2). In addition, recovery of active growth was observed in the six 10 mL cultures after two successive inoculations of liquid nitrogen-frozen cultures of immature colonies of A. gubernaculifera strain NIES-4017 using 6% DMF (Table 2). However, 0% MPN survival and partial recovery of active growth in six 10 mL cultures after two successive inoculations were observed in samples of mature colonies of A. gubernaculifera strain NIES-4017 treated with DMF or HA (Table 2). In contrast, < 0.1% MPN survival was found in samples of A. perforata strain NIES-564 treated with 3% HA, with mature or immature colonies (Table 2). The highest rate of MPN survival among the eight cryogenic treatments in A. perforata strain NIES-564 was 5.5 ± 5.9% when mature colonies were mixed with 3% HA during two-step freezing (Table 2).

Because the effect of sample parameters (colony maturation and cryoprotectants) on recovery after cryopreservation were species-specific, recovery based on the MPN method and two successive inoculations in 10 mL of new medium after the cryopreservation of seven other strains of Astrephomene was examined using immature colonies of A. gubernaculifera with 6% DMF, or mature colonies of A. perforata with 3% HA. Based on these potentially optimized cryopreserved conditions for each species (Table 2), we obtained ≥0.1% MPN viability rates and active growth based on two successive inoculations in 10 mL cultures of A. gubernaculifera strains NIES-418 and NIES-853, and A. perforata strain NIES-565 (Table 3). However, the other four strains of A. gubernaculifera did not recover after freezing in liquid nitrogen and thawing (≥ 0.1% MPN viability), and did not grow after one and two inoculations to 10 mL of medium (Table 3). Thus, five cryopreserved strains of Astrephomene were deposited in the MCC-NIES.

Table 3 Comparison of recovery results of seven strains of two Astrephomene species (Table 1) after possible optimal cryogenic treatment (immature colonies of A. gubernaculifera [AG] with 6% DMF, or mature colonies of A. perforata [AP] with 3% HA; Table 2) in liquid nitrogen
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Discussion

Because Astrephomene requires organic compounds such as acetate for photoheterotrophy, and grows extremely rapidly under photoheterotrophic conditions [14] (Additional file 1: Fig. S2; Additional file 2: Text S1), serial inoculations of living cells to new media during short intervals are required for maintenance of living cultures [10, 16] (https://mcc.nies.go.jp/index_en.html). In addition, during the long-term maintenance of growing cultures by subculturing, the ability to perform normal morphogenesis gradually decreases in Astrephomene [10]. Thus, cryopreservation of culture strains of Astrephomene is needed.

In the present study, we determined the optimal liquid-nitrogen cryopreservation conditions for A. gubernaculifera strain NIES-4017 and A. perforata strain NIES-564 by selecting mature or immature colonies of Astrephomene and two types of cryoprotectants, DMF and HA (Table 2). Amidic and acetonic cryoprotectants, such as DMF and HA, enable cryopreservation of cells based on their ability to cross the cell membrane and cytotoxic effects [19]. We examined MPN survival of unfrozen cells of mature and immature colonies of two species of Astrephomene treated with 3% DMF, 6% DMF, 3% HA and 6% HA (Additional file 1: Table S1, Fig. S3). When immature colonies were treated with 6% DMF, unfrozen cells of A. gubernaculifera strain NIES-4017 exhibited a moderate survival rate (39%), but frozen NIES-4017 cells showed the highest survival rate (11%) among all frozen cell types. By contrast, a high survival rate (99%) for unfrozen cells and a low rate (0.032%) for frozen cells were observed with A. perforata strain NIES-564 (Table 2; Additional file 1: Table S1, Fig. S3). Using mature colonies treated with 3% HA, > 100% survival was detected for unfrozen cells of A. gubernaculifera, compared to 0% for frozen colonies (Additional file 1: Table S1, Fig. S3). By contrast, mature colonies of A. perforata treated with 3% HA had the highest survival rate (5.5%) among frozen cell types and a moderate survival rate (57%) relative to the other unfrozen cell types (Table 2; Additional file 1: Table S1, Fig. S3). Therefore, the ability of HA and DMF to cross the cell membrane, and their toxic effects on cells in immature and mature colonies, differ between A. gubernaculifera and A. perforata.

In A. gubernaculifera strain NIES-4017, mature colonies treated with 3% DMF, 6% DMF and 3% HA exhibited 0% MPN survival after freezing and thawing. By contrast, immature colonies showed a < 0.2% MPN survival rate when treated with 3% DMF, 6% DMF, or 3% HA (Table 2). The difference in survival between immature and mature colonies of A. gubernaculifera could be attributed to differences in cell volume. Mature colonies of Astrephomene contain larger cells than immature colonies (Fig. 2A, B). Cell size is a critical factor for cryopreservation; cryopreserving large algal cells is problematic [26, 27]. However, in A. perforata strain NIES-564, mature colonies treated with 3% HA showed the highest MPN survival rate (5%) after freezing and thawing, while immature colonies treated with 3% HA had a 2.2% MPN survival rate (Table 2). Therefore, cell size may not critically influence the survival of A. perforata cells.

A. gubernaculifera strain NIES-4017 showed 11% MPN survival when immature colonies were treated with 6% DMF (Table 2). However, the four other strains of A. gubernaculifera showed < 0.1% MPN survival when immature colonies were treated with 6% DMF (Table 3). These A. gubernaculifera strains have been maintained by serial inoculations in liquid cultures since their establishment [16] (https://mcc.nies.go.jp/index_en.html). A. gubernaculifera strain NIES-4017 was originally established in 2014 from a single colony in a re-wetted soil sample [10], whereas other strains of this species were established from 1962 to 1981 (Table 1). During the cryopreservation of vegetative colonies of Gonium pectorale, 6% DMF as a cryoprotectant in two-step freezing was effective for cryopreservation, with MPN survival rates of ≥0.1% being maintained in 10 strains from the MCC-NIES [20]. However, three other strains of G. pectorale did not exhibit MPN survival rates ≥0.1% under identical cryogenic conditions (6% DMF) [20]. These three strains (NIES-2261, 469 and 570) have been maintained as growing subcultures since establishment of the original cultures in the period 1979–1994 [20]. Therefore, long-term maintenance of algal strains as growing subcultures by serial inoculation could decrease the survival rates of some strains of Gonium and Astrephomene.

Conclusion

A. gubernaculifera colony maturation and cell volume are critical factors affecting survival after cryopreservation, possibly as a result of cryoprotectant permeability and/or toxicity (Additional file 1: Fig. S3). Large reproductive cells in mature colonies of A. gubernaculifera (Fig. 2B) do not survive 6% DMF treatment, which enables cryopreservation of small reproductive cells (Fig. 2A) (Table 2). Although this factor is not critical in A. perforata and may be species-specific (Table 2), the selection of cells of a suitable age or size may be important for successful cryopreservation in other colonial or multicellular volvocine genera.

Cryopreservation of some long-term-maintained strains of A. gubernaculifera (Table 3) and G. pectorale (NIES-2261, 469 and 570) is difficult [19]. However, strains established concomitantly are readily cryopreserved, particularly of complementary mating types of G. pectorale (NIES-2262, 468, and 569, respectively) [20]. Thus, during the long-term maintenance of cultures by subculturing, survival after cryopreservation may be decreased in certain strains of multicellular volvocine algae. Similarly, the inducibility of sexual reproduction and ability to perform normal morphogenesis gradually decrease during the long-term maintenance of cultures of multicellular volvocine species [10, 28]. Therefore, cryopreservation of newly established culture strains is important for future studies of multicellular volvocine algae.

The present study demonstrated that two species of Astrephomene can be cryopreserved using the optimal cryopreserved conditions for each species (Table 2). However, the survival rates are still low [11 ± 13% (0.36–33%) in A. gubernaculifera strain NIES-4017 and 5.5 ± 5.9% (0.12-12%) in A. perforata strain NIES-564 (Table 2)], which highlights that more effective conditions need to be standardized to obtain better survival.