PULSED ELECTRIC FIELD TREATMENT OF BIOLOGICAL CELLS

Abstract

The present invention relates to methods of increasing metabolic activity and stimulating cell proliferation of biological cells through the use of pulsed electric field (PEF) treatment, especially through the use of nanosecond pulsed electric field (nsPEF) treatment.

Claims

1. An in vitro method of increasing metabolic activity of biological cells, said method comprising the following steps: (a) suspending biological cells in an electrically conductive liquid, thereby forming a suspension, (b) positioning the suspension between two electrodes, and (c) applying from 1 to 100 pulses, preferably from 2 to 30 pulses, of electricity between the electrodes, thereby increasing metabolic activity of the biological cells; characterized in that a voltage increase between the two electrodes from 10% to 90% of a target voltage of the pulses of electricity takes place within a time period of 0.1 to 100 ns, preferably within a time period of 1 ns to 25 ns; wherein the pulses of electricity have a pulse duration of between 5 to 5000 ns, preferably of between 50 ns to 300 ns; and wherein the pulses of electricity, when reaching the target voltage, have an electric field strength of 0.5 kV/cm to 50 kV/cm, preferably of 1.0 kV/cm to 30 kV/cm.

2. The method of claim 1, wherein the biological cells are selected from the group consisting of bacterial cells, yeast cells, fungal cells, microalgae cells, plant cells, animal cells, and human cells.

3. An in vitro method of stimulating cell proliferation and/or increasing metabolic activity of biological cells, said method comprising the following steps: (a) suspending biological cells in an electrically conductive liquid, thereby forming a suspension, (b) positioning the suspension between two electrodes, and (c) applying from 1 to 100 pulses, preferably from 2 to 30 pulses, of electricity between the electrodes, thereby stimulating cell proliferation; characterized in that a voltage increase between the two electrodes from 10% to 90% of a target voltage of the pulses of electricity takes place within a time period of 0.1 to 100 ns, preferably within a time period of 1 ns to 25 ns; wherein the pulses of electricity have a pulse duration of between 5 to 5000 ns, preferably of between 50 ns to 300 ns; and wherein the pulses of electricity, when reaching the target voltage, have an electric field strength of 0.5 kV/cm to 50 kV/cm, preferably of 1.0 kV/cm to 30 kV/cm; wherein the biological cells are selected from the group consisting of bacterial cells, yeast cells, fungal cells, microalgae cells, plant cells, and animal cells, wherein the animal cells are selected from the group consisting of Spodoptera frugiperda and Trichoplusia ni.

4. The method of claim 2, wherein the bacterial cells are selected from the group consisting of Escherichia coli and Corynebacterium glutamicum; the yeast cells are selected from the group consisting of Pichia pastoris and Saccharomyces cerevisiae; the microalgae cells are selected from the group consisting of Galdieria sulphuraria and Aurantiochytrium limacinum; the plant cells are selected from the group consisting of Hordeum vulgare and Oryza sativa; and the animal cells are selected from the group consisting of Spodoptera frugiperda and Trichoplusia ni.

5. The method of claim 1, wherein step (c) is carried out in a recirculation process.

6. The method of claim 1, wherein step (c) is carried out in a batch process.

7. An in vitro method of stimulating cell proliferation and/or increasing metabolic activity of biological cells, said method comprising the following steps: (a) suspending biological cells in an electrically conductive liquid, thereby forming a suspension, (b) positioning the suspension between two electrodes, and (c) applying from 1 to 100 pulses, preferably from 2 to 30 pulses, of electricity between the electrodes, thereby stimulating cell proliferation; characterized in that a voltage increase between the two electrodes from 10% to 90% of a target voltage of the pulses of electricity takes place within a time period of 0.1 to 100 ns, preferably within a time period of 1 ns to 25 ns; wherein the pulses of electricity have a pulse duration of between 5 to 5000 ns, preferably of between 50 ns to 300 ns; and wherein the pulses of electricity, when reaching the target voltage, have an electric field strength of 0.5 kV/cm to 50 kV/cm, preferably of 1.0 kV/cm to 30 kV/cm; wherein step (c) is carried out in a recirculation process.

8. The method of claim 7, wherein the biological cells are selected from the group consisting of bacterial cells, yeast cells, fungal cells, microalgae cells, plant cells, animal cells, and human cells.

9. The method of claim 8, wherein the bacterial cells are selected from the group consisting of Escherichia coli and Corynebacterium glutamicum; the yeast cells are selected from the group consisting of Pichia pastoris and Saccharomyces cerevisiae; the microalgae cells are selected from the group consisting of Galdieria sulphuraria and Aurantiochytrium limacinum; the plant cells are selected from the group consisting of Hordeum vulgare and Oryza sativa; and the animal cells are selected from the group consisting of Spodoptera frugiperda and Trichoplusia ni.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0094] FIG. 1.: Metabolic activity of different E. coli samples determined by FDA (fluorescein diacetate) cleavage. The E. coli cell samples received different numbers of electric pulses of a nanosecond pulsed electric field (nsPEF) treatment in a recirculation process. All E. coli cell samples exhibited an increase in metabolic activity due to the nsPEF treatment.

EXAMPLES

Example 1

[0095] A twin bioreactor system was used for parallel cultivations. The parallel cultivations were inoculated with the same amount of pre-culture from the same feed train of the selected bacterial cell Escherichia coli. The reactor cultivations as well as the pre-culture/inoculum were performed in the modified, defined MD-FB medium: [0096] 14.3 g L.sup.?1 (pre-culture) and 28.7 g L.sup.?1 glycerol 85%, [0097] K.sub.2HPO.sub.4 2.70 g L.sup.?1, [0098] KH.sub.2PO.sub.4 13.2 g L.sup.?1, [0099] NaCl 2.04 g L.sup.?1, [0100] (NH.sub.4)SO.sub.4 4.1 g L.sup.?1, [0101] Antifoam 205 0.05 g L.sup.?1, [0102] ddH.sub.2O 920.8 g L.sup.?1, [0103] 0.8 mL MgSO.sub.4*7 H.sub.2O stock (300 g L.sup.?1), [0104] 10.0 mL Fe(III) citrate stock (10.0 g L.sup.?1), [0105] 10 mL Na-EDTA*2 H.sub.2O stock (0.84 g L.sup.?1), [0106] 2.8 mL Thiamine HCl stock (45.0 g L.sup.?1), [0107] 2.9 mL TE stock (COCl.sub.2*6 H.sub.2O 0.16 g L.sup.?1, [0108] MnCl.sub.2*4 H.sub.2O 1.42 g L.sup.?1, [0109] H.sub.3BO.sub.3 0.01 g L.sup.?1, [0110] Na.sub.2MoO.sub.4*2 H.sub.2O 0.02 g L.sup.?1, [0111] CaCl.sub.2)*2 H.sub.2O 1.44 g L.sup.?1, [0112] AlCl.sub.3*6 H.sub.2O 0.04 g L.sup.?1, [0113] ZnSO.sub.4*7 H.sub.2O 0.87 g L.sup.1, [0114] CuSO.sub.4*5 H.sub.2O 1.55 g L.sup.?1, [0115] NiCl.sub.2*6 H.sub.2O 0.01 g L.sup.?1

[0116] Cultivation conditions are summarized in Table 1:

TABLE-US-00001 TABLE 1 Parameter Value Unit Reactor working volume 10 [L] pH 7 [] Aeration (sterile air) 3 [vvm] Temperature 30.0 [? C.] Pressure 0.5 [bar] Stirrer 1200 [rpm]

[0117] Upon treatment the metabolic activity was expressed as the rate and total amount of individual cellular FDA (fluorescein diacetate) cleavage. Standard staining protocols were used in the experiments and analysed in a flow cytometer (Ehgartner D, Herwig C, Neutsch L. At-line determination of spore inoculum quality in Penicillium chrysogenum bioprocesses. Appl Microbiol Biotechnol. 2016; 100(12):5363-73; S?derstr?m BE. Vital staining of fungi in pure cultures and in soil with fluorescein diacetate. Soil Biol Biochem. 1977; 9:59-63; and Pekarsky, A., Veiter, L., Rajamanickam, V. et al. Production of a recombinant peroxidase in different glyco-engineered Pichia pastoris strains: a morphological and physiological comparison. Microb Cell Fact 17, 183 (2018)).

[0118] Table 2 summarizes the nsPEF conditions for the control and five different samples.

TABLE-US-00002 TABLE 2 Sample No. 010 012 016 018 019 0112 Potential difference [kV] Control 10 10 10 10 10 Pulse length [ns] 100 100 100 100 100 Pulses of electricity per 4 6 8 10 12 treatment passage [] Electrode distance [cm] 0.5 0.5 0.5 0.5 0.5 0.5

[0119] An increase in metabolic activity was detected for all treated samples (see FIG. 1). Surprisingly, the greatest increase of metabolic activity was observed for sample no. 12, which received the lowest number of pulses (i.e. 4 pulses). Sample No. 012 exhibited an increase of metabolic activity as compared to control sample 010 from 90 FIU to 120 FIU, which corresponds to an increase of about 30%

Example 2

[0120] A twin bioreactor system was used for parallel cultivations. The parallel cultivations were inoculated with the same amount of pre-culture from the same feed train of selected yeast cell Pichia pastoris. The reactor cultivations were performed in the modified, defined medium (Hellwig et al., 2001): [0121] 35.29 g L.sup.?1 Glycerol 85%, [0122] K.sub.2SO.sub.4 2.86 g L.sup.?1, [0123] KOH 0.64 g L.sup.?1, [0124] MgSO.sub.4*7 H.sub.2O 2.32 g L.sup.?1, [0125] CaSO.sub.4*2 H.sub.2O 0.17 g L.sup.?1, [0126] Na-EDTA*2 H.sub.2O 0.6 g L.sup.?1, [0127] NaCl 0.22 g L.sup.?1, [0128] Antifoam 205 0.1 g L.sup.?1, [0129] H.sub.3PO.sub.4 (85% 7.19 g L.sup.?1, [0130] Water 860 g L.sup.?1, [0131] PTM1 stock 4.35 mL L.sup.?1 (CuSO.sub.4 6.0 g L.sup.?1, NaI 0.08 g L.sup.?1, MnSO.sub.4*H.sub.2O 3.0 g L.sup.?1, Na.sub.2MoO.sub.4*2 H.sub.2O 0.2 g L.sup.?1, CoCl.sub.2*6 H.sub.2O 0.5 g L.sup.?1, ZnCl.sub.2 20.0 g L.sup.?1, FeSO.sub.4*7 H.sub.2O 65.0 g L.sup.?1, H.sub.2SO.sub.4 (98%) 50 mL l.sup.?1, H.sub.3BO.sub.3 0.02 g L.sup.?1), [0132] Biotin stock 500?4.35 mL L.sup.?1 (0.2 g L.sup.?1)

[0133] Cultivation conditions are summarized in Table 3:

TABLE-US-00003 TABLE 3 Parameter Value Unit Reactor working volume 10 [L] pH 7 [] Aeration (sterile air) 3 [vvm] Temperature 30.0 [? C.] Pressure 0.5 [bar] Stirrer 800 [rpm]

[0134] Upon treatment the metabolic activity was increased on an individual cell basis, based on the analysed parameters.

[0135] Table 4 summarizes the nsPEF conditions for the control and four different samples.

TABLE-US-00004 TABLE 4 Sample No. 0 1 2 3 4 Potential difference [kV] Control 5 5 5 5 Pulse length [ns] 100 100 100 100 Pulses of electricity per 2 3 4 6 treatment passage [] Electrode distance [cm] 0.5 0.5 0.5 0.5 0.5