Electropulsing of acinar cells isolated from rat pancreas

97

Bioefectrochemistty and Eioenergetics, 26 (1991) 97-106 A section of J. Eiectroanal. Chent., and constituting Vol. 321 (1991) Elsevier Sequoia S.A., Lausanne

Electropulsing of acinar cells isolated from rat pancreas Studies of reversible membrane perforation and blebbing H. Kosowski and G. Letko * Division of Experimental Surgery. Clinic of Surgev. Medical Academy of Magdehurg. Leipriger Str. 44, O-3090 Magdeburg (Germany) (Received

19 September

1990;

in revised form 22 February

1991)

Abstract Electropulsing of pancreas cells was performed by the charge pulse technique. Injury to the cells and their repair were determined by means of trypan blue exclusion. The voltage and the capacitance were selected such that membrane perforation and membrane deformation (as blebs) in standard cells (cells with a survival rate in excess of 92% after 4 h) was reversible. Depending on the electrical conditions, the percentage of cells without membrane perforation one minute after electropulsing ranged from 15% to 62%. and the percentage of cells without membrane deformation ranged from 26% to 51%. Irrespective of the electrical parameters, repair of membrane perforation was complete after 15 min. In contrast, 30 min to 2 11were required for repair of blebs to finish. Identical investigations were conducted with injured cells. The percentages after electropulsing were higher than those for standard celh. No repair was observed. Injury and repair were found to also depend on the functionality of cells. Comparison between standard cells and the cells to be studied permits the functiynahty of such celk to be assessed_

INTRODUCTION

Permeabilization of biological cells permits the investigation of cell structures and cell processes as well as manipulation of cells. The methods used depend on the objective. Chemical and biological methods of permeabilization [l] are facile, but result mainly in irreversibly damaged cells. ____ _ In contrast, electropulsing [2,3] causes reversible permeabilization of bio!ogical cells. * To whom correspondence should be addressed. 03024598/91/%03.50

0 1991 - Elsevier Sequoia S.A.

98

Electropulsing of cells with the aim of a reversible membrane perforation [4] has been in common use for various purposes for some time [4-91. Such reversible injury permits cell fusion [4,5] or provides access for high-molecular materials to the cell interior [7]. Moreover, this approach can be adapted to determine cell parameters through electrical variables with little or no damage to the cells [6,10]. Cell deformations are another form of injury [ll], Also known as blebs, such deformations typically result from external factors such as ischaemia [12] and hypoxia [13]. Blebs are not necessarily a characteristic of cell death, since repair of blebs is possilole [14]. Assessing the viability of isolated cells is essential to cell physiology and pathophysiological investigations. The trypan blue exclusion method (TET) is the most common viability test. Cells with an intact plasma membrane are not stained by the dye, whereas stained cells are regarded as dead. This approach is disadvantageous in that it takes several hours before even minute alterations are detected as measurable cell staining. The sensitivity of TET has been improved by considering the time course of the diminishing cell count over a longer period (e.g., 4 hours). Yet, it is desirable to obtain quick information on the relevance of isolated cells. Therefore, we investigated whether or not the behaviour of isolated cells after electropulsing provides information on the functionality of those cells. It was the aim of this paper to determine the interdependence between cell injury and repair, and the experimental conditions, such ai the discharge voltage and capacitance, so as to ascertain the functionality of cells. EXPERIMENTAL

Acinar cells separated from rat pancreas by collagenase treatment 115,161 were used throughout. The cell suspension had an average cell density of 20 X lo6 cells/ml and was transferred to an Erlenmeyer flask, incubated with oxygen, and kept in a shaking incubator at 37*C. Two samples from each of the suspensions studied were tested. The first sample contained cells were not subjected to electropulsing and was kept in the shaking incubator at 37°C throughout the period of investigation. Upon completion of cell isolation and 1, 2, 3 or 4 hours later, the trypan blue negative cells (TN) CT, - CT, were counted in a Neubauer chamber by TET, and then the cell density was determined. If after 4 hours the survival rate SR, = CT,1oO%/cT, was in excess of 92%, the cells. _of, that particular suspension were classed as standard cells. Suspensions not containing TN after 3 hours (SR, = 0) were classed as having injured cells. The second sample was used to study the influence of exposure to electropulsing. The latter was applied by means of a charge pulse technique 117,181in a discharge chamber consisting of a 1 ml glass syringe with metal bottom and metal piston used as electrodes. Specifications of the sample chamber were: 1 ml glass syringe (Injecta, Jena, Germany); Separation of electrodes 8 mm (distance between syringe bottom

Fig. 1. Classification of cells wiih trypan blue added. T?JC: Txypan blue negative cells without blebs; TPC: Trypan blue positive cells without blebs; TNB: Trypan blue negative blebs; TPB: Try-pan blue positive blebs; TN: Trypan blue negative cells; TP: Trypan blue positive cells; NB: non-blebs; BS: blebs. The arrows indicate possible changes in the cell forms.

and piston); piston area 65 mm*; volume of sample chamber 0.52 ml. For standard cells, the discharge voltage was 1.6 kV and 2.4 kV and the capacitance 0.25 PF aud 1.0 pF, the respective figures for the izjured cells being 2.4 kV and 0.25 FF. Thirty minutes after completion of cell isolation, the sample chamber was filled with the cell suspension free from air bubbles and exposed to the discharge voltage. Then, the cell suspension was transferred to a 5 ml plastic vessel, incubated with oxygen and kept in a shaking incubator for 60 min at 23°C. This low temperature was necessary to ensure a slow repair process [6,19], and afterwards to permit cell counting by TET. Once repair was completes after some 60 min, the sample was stored in a shaking incubator at 37°C to establish temperature conditions identical to those for the first sample. The four cell forms (Fig. 1) were counted before and 1, 5,10,15, 30,60,120,180 and 240 min after electropulsing, in the Neubauer chamber by means of TET, where: TNC are Trypan blue negative cells without blebs; TPC are Trypan blue positive cells without blebs; TNB are Trypan blue negative blebs, and TPB are Trypan blue positive blebs. These cell forms can be classed into two groups. The first group comprising. TN = TCN + TNB (trypan blue negative cells) and TP = TPC + TPB (trypan blue positive cells) from the information on membrane perforation and repair, and the second group comprising NB = TNC + TPC (non-blebs) and BS = TNB + TPB (blebs) from the information on membrane deformation and repairThe results were obtained from more than four tests (n > 4) and are given as the mean f the standard deviation.

.

.

Fig. 2. Suspension of pancreas cells. (a) Before electropulsing. (B) One minute after electropulsing, electrical conditions: 2.4 kV; 0.25 PF. The high percentages of TPC and TPB are the result of electropulsing. (C) Thirty minutes after electropuIsing, electrical conditions are the same as for (B). The distinct cell deformations (TNB) are the result of electropulsing.

The total cell count before electropulsing SC0

=TNo+TPO=NB,+BSo

was used as a reference for all cell counts in a test. RESULT’S AND

DISCUSSION

Suspension of pancreas cells without electropulsing is shown in Fig. 2A. TPC and TBC in Fig. 2B were produced by electropulsing. Distinct deformations (TNB in Fig. 2C) are formed after membrane perforation. These deformations are repaired in the course of time or are the early stage of an onsetting cell lysis. Standard cells not exposed to electropulsing

The TN of standard cells not exposed to electropulsing (Fig. 3a) had an average initial injury of 7% and a survival rate of 92%. The percent NB (Fig. 4a) dropped

34-c

23’ C

0

60

120

180

220

Time /min

Fig. 3. Time course of trypan blue negative ceils (TN) of standard cells. Values are related to the initial total cell count. Cells without electropulsing observed after incubation at 37OC, electropul.sed cells observed after dectropulsing at 23OC during the first hour, and at 37% during the next 3 h. Relationship e) without between symbols, electrical conditions (U, C). and number of tests (n): (a, e----A) 2.4 kV, 0.25 PF, 7; (d, O--*--O) 2.4 0) 1.6 kV, 0.25 pF, 5; (c. A-electropulsing, 9; (b, OkV, 1.0 FF, 5.

steadily from 85% to 81%. 240 minutes later, the total cell count (Fig. Sa) had dropped to 93W, i.e., cell lysis had set in for 7% of the cells. Cell suspensions showing these percentages were classed as standard cells of consistent functionality. Standard cells exposed to electrqulsing For the charge pulse technique employed, the voltages applied were l-6 kV and 2.4 kv, and the capacitances 0.25 PF and 1.0 PF. These values correspond to a field strengths of 2 kv/cm and 3 kv/cm. The time constants were about 25 ps and 100 ~LS,this is associated with a pulse width time of about 75 ps and 300 ps, respectively. They ranged from 2 kV/cm to 20 kV/cm as well as from nanoseconds to milliseconds for a reversible membrane perforation as reported previously by others such as Zimmermann et al. [9].

Temperature

1

0

t

I

I

30

I

1

’ I_ 60

120

180

240

Time / min Fig. 4. ‘Time course of non-bIebs (NB) of standard cek The temperature conditions, the electrical conditions, and the number of tesis are the same as for Fig. 3.

the symbols

used,

The time course of TN exposed to electropulsing by varied voltage and capaci- . tance is shown in Fig. 3. One minute aft& electropulsing the 63% TN at 1.6 kV (Fig. 3b) the 34% TN at 2.4 kV and 0.25 PF (Fig. 3c), and the 14% TN at 2.4 kV and 1.0 FF (Fig. 3d), reflect the differing effects of field strength and time constant on the cell suspension 141. Further evidence is obtained from the time course of repair. In all t- * three tests, repair of TP to TN was complete after 15 minutes. However, the same repair time in all tests implies that the electrical conditions have no effect on repair. Cell proportions which influence the repair of membrane perforation are not altered by exposure to a breakdown voltage, even though other irreversible mechanical damage to the membranes may occur [20]. The time course of NB exposed to varied voltage and capacitance is shown in Fig. 4. One minute after electropulsing the 51% NB at 1.6 kV (Fig. 4b), the 35% NB at 2.4 kV and 0.25 PF (Fig. 4c), and 26% NB at 2.4 kV and 1.0 J.LF(Fig. 4d), also reflect the differing effects of field strength and time constant on rhe cell suspension. As compared with TN, more time was required for repairing NB. Nevertheless, Fig. 4 reflected a marked dependence of the repair of BS to NB, on the nature of the

103 Temperature

t 0

t

I

I

30

I

1

’ I-, 60

120

180

260

Time / min Fig. 5. Time course of total cell count of standard cells. The temperature conditions, the electrical conditions, and the number of tests are the same as for Fig. 3.

the symbols (a-d),

electrical conditions. The repair of NB was complete after 30 minutes at 2.4 kV and 0.25 PF (Fig. 4c). In contrast, at 1.6 kV and 0.25 FF (Fig. 4b) a slight, but permanent repair occurred over the entire 240 minutes. Distinct deformation of the time course of repair occurs at 2.4 kV and 1.0 PF (Fig. 4d). Repair began as much as 20 minutes after electropulsing. After 60 minutes electropulsing, repair was accelerated owing to the higher temperature [6,19]. This is not only attributable to the dependence on the electrical field strength and the time constant. The electrical energy U2C/2 which is converted to heat in the cell suspension, does exert an effect on membrane deformation. After 60 minutes electropulsing the cell suspension contained 14% TP and 35% BS at 2.4 kV and 1.0 pF, which represents the difference between the total cell wunt (Fig. 5d) and TN (Fig. 3d), or NB (Fig. 4d). These percentages_yere higher than those of the cells not exposed to electropulsing. Consequently, not ail of TP and BS were repaired to TN and NB. The marked decrease in TN and NB levels is attributable to the electropulsing. Injured cells

Figures 6 and 7 show the time course of cells injured during isolation. Those cells which were not exposed to electropulsing, were dead within 3 h (SR, = 0). After

104 Temperature

37’C

80

3

I i

30

60

120

180

240

Time / tnin

Fig. 6. Time course of total cell count and TN of injured cells (n = 5) in relation to the standard cells under temperature conditions identical with those in Fig. 3. Electrical conditions: U= 2.4 kV; C = 0.25 pF. (a, OS- s-.0) total cell count of injured cells; (b, Oo) TN of injured cells; (c, I --1) total cell count from standard cells (Fig. 5~): (d, 9- -----a) TN from standard ceils (Fig. 3~).

electropulsing the comparison with TN (Fig. 6d) and NB (Fig. 7b) of the standard cells showed higher TN (Fig. 6b) and NB (Fig. 7a) levels percentages of 59% and 52%, respectively. No repair was observed. The continuous decrease in the total cell count (Fig. 6a) was another unique feature, with cell. iysis setting occurring continuously, a phenomenon not observed for the standard cells. CONCLUSIONS

The charts (Figs. 3-5) display reversible membrane perforation and membrane deformation (blebs) after electropuking within the set voltage and capacitance limits, provided.standard cells are used. These are cells having a good functionality. Differences as to the amount of injury and the time course of repair are indicative of altered functionality of the cells. Cells with an unknown functionality can be assessed by comparing their injury and repair with those for standard cells, while using identical experimental condi-

105 Temperature 23-C

0

37-c

I I

60

30 Time

1

120

180

240

/ min

Fig. 7. Time course of NB of injured cells in relationship to the NB of standard cells under temperature conditions identical with those in Fig. 3, and electrical conditions identical with those in Fig. 5. (a. A--A) NB of injured celis; (b, A------ A) NB of standard cells (Fig. 4c).

tions, viz., discharge voltage, capacitance, sample chamber, and temperature. These should be chosen for the particular standard cells. Such conditions are optimal, when, after exposure to electropulsing, the percentages of TN and TB are low ( < 40% of the total cell count), while high percentages of TP and BS are repaired to TN and NB. In the present study, optimal discharge voltage and capacitance figures were 2.4 kV and 0.25 pF, respectively (Figs. 3-5). Lower voltages produced less TN and NB, Higher capacitance levels impaired the repair of BS to NB. Cells having a dissimilar functionality, e.g., injured cells (Figs. 6 and 7), exhibited a different percentage of injured cells and an altered time course of rep& after eiectropulsing. Changes in TN and NB percentages as well as in repair of TP and BS to TN and NB are indicative of an altered functionality, thus enabling cell changes to be interpreted.

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