Cryoconservation Modifies Ion Transport Pathways in the Skin Microenvironment: An In Vitro Study

Abstract

Due to the lack of skin donors, the short time frame for conducting the procedure, and the increasing demand for tissue specimens, the proper storage conditions for skin fragments have gained critical importance. Therefore, the search for methods for storing skin tissue long-term, ensuring its physiological functions, is a matter of considerable interest. Freezing skin fragments in a cryoprotectant solution, such as dimethylsulfoxide (DMSO), can be a valuable complement to tissues for transplantation and for supplying difficult-to-heal wounds. This study aimed to assess the effect of deep freezing rabbit skin fragments immersed in a 5% DMSO solution on their electrophysiological parameters. Control (n = 23) and defrosted skin specimens were incubated in Ringer (n = 21), amiloride (n = 26), and bumetanide (n = 24) solutions. Then, resistance (R), potential difference (PD), and minimal and maximal PD were measured. The specimens did not show differences in R values compared to controls, which means that the skin subjected to freezing was compact and durable. However, the tissue subjected to freezing in DMSO solution presented increased transport of sodium and chloride ions, which may translate into a change in pain perception, the development of hypersensitivity and/or allergy, and the initiation of repair and regeneration processes.

1. Introduction

Appropriately treating difficult-to-heal wounds is a challenge for modern medicine [1,2,3,4]. The removal of damaged tissue, followed by the transplantation of healthy skin fragments or the application of unaltered tissue and/or skin substitutes to the site of a lesion, is used as a method to support healing [1,3,5]. The human skin is an organ that constitutes an environment for multiple physiological and biochemical processes and contributes to maintaining homeostasis in the body [6,7]. Therefore, there is an increasing demand for skin fragments for the above-mentioned procedures [3,5]. Due to the deficiency of organ donors, including skin donors, the short time frame to conduct the procedure, and increasing demand for tissue specimens, the proper storage conditions for skin fragments have gained critical importance [1,2,3,5].

The leading cause of failure in freezing tissue fragments is damage to cells by water crystals; therefore, a procedure involving immersing tissue samples in solutions with cryoprotective properties has been established, usually involving the use of dimethyl sulfoxide (DMSO) or glycerol, which are dehydrating substances that do not affect the cell structure [2,4,5,8,9]. In recent years, additional attention has been paid to maintaining the physiological properties of microspaces in frozen tissue specimens [10,11,12,13]. Changing osmolality in the intercellular spaces results in modifications of the properties of proteins and the activity of enzymes operating in this specific microenvironment [12,13]. Additionally, maintaining proper hydration and ion concentration in the extracellular matrix is crucial to ensure the adequate growth, development, and differentiation of keratinocytes [11] and fibroblasts [10].

Intense transport of sodium and chloride ions occurs within the skin [14,15,16], which generates a local electric field [15,16]. Apically, the main route of Na⁺ adsorption is the epithelial sodium channel (ENaC) [17,18,19], while the secretion of chloride ions occurs via cystic fibrosis transmembrane conductance regulator (CFTR) [20]. Coordinated transport of sodium and chloride ions is conducted by Na-K-Cl cotransporter (NKCC) and the sodium-potassium pump [17,18,21]. The overlap between these processes generates and maintains the difference in transepithelial electrical potential, which can be measured in an Ussing chamber due to ion flow both in stationary conditions and during mechanical or mechanical–chemical stimulation [14,15,22]. Transepithelial electrical resistance can also be measured using this method. Changes in ion transport result in water flow into or out of the extracellular space, changes in cell reactivity, and the ability to release and receive signaling particles [8,17,18,20,23]. Changes in the routes of transport of sodium and/or chloride ions can be associated with changes in the shape and/or movement of keratinocytes [19], increased nociception [23,24], and the migration of immunocompetent cells [17], which can lead to wound healing difficulties [19,25], hyperreactivity [15], and the development of hypersensitivity and/or allergies [20]. Additionally, maintaining a directed electric field around cells is associated with releasing chemotactic substances that initiate and support wound healing [15].

Changes in ion transport in the skin resulting from deep freezing have not been analyzed [4,8,16,26]. The present study aimed to assess the changes in ion transport occurring in the skin after freezing it at −80 degrees using DMSO as a cryoprotectant. Changes in ion transport are measured as transepithelial electrical resistance and transepithelial electrical potential under stationary conditions and during mechanical and mechanical–chemical stimulation to evaluate changes in the transport of sodium and chloride ions in full-thickness skin fragments subjected to freezing at −80 °C. In light of the globally increasing number of cases of skin fragment transplantation, we believe that determining the mechanisms of ion transport in skin fragments stored in low-temperature conditions is essential to clarify the pathomechanisms of adverse reactions occurring in transplanted fragments and to prevent negative impacts on patient health [2,3,5].

2. Materials and Methods

The experiment was conducted in a modified Ussing chamber, which measured electrophysiological parameters of the epithelial tissue according to the method formerly used by the authors [17,27].

2.1. Animals

The experiment was conducted using isolated skin fragments taken from 9 albino New Zealand rabbits of both sexes aged 2–3 months, weighing 3.5–4.0 kg, after their deaths. The animals were euthanized by carbon dioxide asphyxiation, and their deaths were confirmed by a qualified person using two methods.

The material was taken using a scalpel from the inner side of the ear; cartilage and muscle were separated. Eight to ten skin fragments were acquired from one skin lobe of the ear. Thus, keratinocytes, corneocytes, fibroblasts, immunocompetent cells, and nerve fiber terminals were present in the obtained skin fragments [7].

2.2. Chemicals and Solutions

The following abbreviations for chemicals and solutions were used in the experiment:

-

RS—the Ringer solution (K⁺ 4.0 mM; Na⁺ 147.2 mM; Ca²⁺ 2.2 mM; Mg²⁺ 2.6 mM; Cl⁻ 160.8 mM), a basic solution with iso-osmotic properties and a pH of 7.4. Mineral compounds (KCl, NaCl, CaCl₂, MgCl₂) were purchased from Avantor, Zabrze, Poland.

-

DMSO—dimethyl sulfoxide 5% (0.704 mol/L) (Sigma-Aldrich, Burlington, NJ, USA) solution, diluted in RS.

-

Ami—amiloride hydrochloride hydrate (0.1 mmol/L) 3,5-diamino-6-chloro-2-carboxylic acid (Sigma-Aldrich, Burlington, NJ, USA); used as an inhibitor of the transepithelial transport of sodium ions, diluted in RS.

-

Bume—bumetanide (0.1 mmol/L) 3-butylamino-4-phenoxy-5-sulfamoylbenzoic acid (Sigma-Aldrich, Burlington, NJ, USA); used as an inhibitor of the transepithelial transport of chloride ions, diluted in RS.

-

AB—a mixture of amiloride (0.1 mmol/L) and bumetanide (0.1 mmol/L) solutions.

2.3. Experimental Procedure

The experiment was conducted on 94 isolated skin fragments, including 71 study samples and 23 control samples. In the case of the study samples, the isolated skin fragments were incubated in 5% DMSO solution for 30 min, in 1 mL of 5% DMSO solution for 2 cm² of skin specimens [4,27]. Subsequently, excess liquid was removed with tissue paper, then the skin fragments were placed in separate tubes, transferred into −80 ± 2 °C conditions with a freezing rate of 1 °C/min, and incubated for 45 days. Organ banks commonly store tissue samples at −80 °C [1,3,5]. Defrosting was carried out in three steps, consisting of the subsequent transfer of the tube to: (1) a freezer at −20 ± 2 °C; (2) a refrigerator at +4 ± 2 °C; and (3) room temperature at 23 ± 2 °C, with each transfer followed by 2 h of incubation. For control specimens, the freezing procedure was omitted.

Then, the thawed skin fragments were transferred into RS and incubated for 60 min. After incubation, all specimens were subdivided into pieces of 2 cm² each. In the case of the control samples, the skin fragments with a surface area of 2 cm² each were immersed in RS and incubated for 30 min. The following experimental groups were distinguished (Figure 1):

(1)

Control (n = 23)—the samples were incubated in RS solution.

(2)

Group subjected to deep freezing, divided into 3 subgroups:

(2.1) RS (n = 21)—the samples were subjected to deep freezing at −80 °C with cryoprotection (5% DMSO solution) and were incubated in RS solution, as a model of undisturbed ion transport.

(2.2) Ami (n = 26)—the samples were subjected to deep freezing at −80 °C with cryoprotection (5% DMSO solution) and were incubated in the Ami solution, as a model of inhibited sodium transport.

(2.3) Bume (n = 24)—the samples were subjected to deep freezing at −80 °C with cryoprotection (5% DMSO solution) and were incubated in the Bume solution, as a model of inhibited chloride transport.

Figure 1. The study design. Abbreviations: RS—iso-osmotic Ringer solution, Ami—amiloride (0.1 mmol/L) solution, Bume—bumetanide (0.1 mmol/L) solution, AB—solution of amiloride (0.1 mmol/L) and bumetanide (0.1 mmol/L), DMSO—dimethyl sulfoxide (5%, 0.704 mol/L) solution, PD—transepithelial potential difference measured under stationary conditions (mV), PDmin—minimal transepithelial potential difference measured during a 15-s stimulation of skin surface (mV), PDmax—maximal transepithelial potential difference measured during a 15-s stimulation of skin surface (mV), R—resistance (Ω × cm²).

Figure 1. The study design. Abbreviations: RS—iso-osmotic Ringer solution, Ami—amiloride (0.1 mmol/L) solution, Bume—bumetanide (0.1 mmol/L) solution, AB—solution of amiloride (0.1 mmol/L) and bumetanide (0.1 mmol/L), DMSO—dimethyl sulfoxide (5%, 0.704 mol/L) solution, PD—transepithelial potential difference measured under stationary conditions (mV), PDmin—minimal transepithelial potential difference measured during a 15-s stimulation of skin surface (mV), PDmax—maximal transepithelial potential difference measured during a 15-s stimulation of skin surface (mV), R—resistance (Ω × cm²).

After the incubation in RS, Bume, or Ami, the tissue samples were placed in an Ussing chamber with a horizontal experimental surface of 1 cm². Next, the proper solution (RS, Bume, or Ami) was injected into the chamber. The experiment lasted 30 min per specimen and was conducted at 25 ± 2 °C. The modified Ussing chamber was equipped with a nozzle connected to a peristaltic pump and placed 3 mm above the tissue sample, which allowed mechanical (RS) and mechanical–chemical (Ami, Bume, AB) stimulation for periods lasting 15 s, with a fluid flow rate of 0.06 mL/s (1.0 mL/15 s).

The experiments involved measuring the following parameters:

(1)

transepithelial electric potential—changes in transepithelial electrical potential measured continuously under stationary conditions (PD, mV),

(2)

minimal and maximal transepithelial electrical potential measured during a 15-s stimulation (PDmin, PDmax, mV),

(3)

transepithelial electrical resistance (R, Ω × cm²), determined by stimulating the tissue sample with a stimulus current intensity of 10 µA for each side of the tested skin specimen. Subsequently, the corresponding voltage change was measured, and resistance was counted according to Ohm’s law.

2.4. Data Analysis

Results were summarized in tables as the median and upper and lower quartiles. A non-parametric distribution of data was obtained. Statistical analysis was conducted with Statistica 11.00 software (StatSoft, Inc., Cracow, Poland). To determine the data distribution, the Kolmogorov–Smirnov test was used with Lilefors corrections. The Wilcoxon test was used to compare data from the same incubation conditions, with the statistical significance level set at p < 0.05. The Mann–Whitney U-test was used to detect significant differences (at p < 0.05) for the different experimental conditions in various groups of tissue samples.

3. Results

Median transepithelial R of the control skin fragments incubated in RS ranged between 5874 and 30,794 Ω × cm² (

Table 1. Values of transepithelial electric potential (PD, mV) of skin surface and resistance (R, Ω × cm²) measured under stationary conditions for the control skin specimens treated with the iso-osmotic Ringer solution (RS) and specimens frozen with cryoprotection and incubated in RS, amiloride (Ami, 0.1 mmol/L), or bumetanide (Bume, 0.1 mmol/L) solutions.

	PD (mV)			R (Ω × cm2)
Incubation	Median	Upper Quartile	Lower Quartile	Median	Upper Quartile	Lower Quartile
Control (n = 23)	−0.27	−0.11	−0.45	11,554	30,794	5874
RS (n = 21)	0	0	−0.14	7955	13,267	3676
Ami (n = 26)	0	0.48	−0.23	3101	6700	2202
Bume (n = 26)	−0.12	−0.29	0	5642	10,379	5119

Abbreviations: n—number of skin specimens.

). The tissue samples subjected to freezing in RS showed an R of 3676, remaining at the physiological level. The observed 30% decrease in resistance for tissues subjected to freezing and incubated in RS and 50% decrease for incubation in Ami for the control should be explained by the change in the permeability of frozen tissues for ions and water, and not by extensive damage to cells and/or intercellular spaces. Despite significant changes in the resistance to 13,267 Ω × cm², for the skin specimens incubated in B, median R values varied from 5119 to 10,379 Ω × cm². Despite a clearly marked decreasing tendency in the study group, no statistically significant differences were found between the RS and Bume groups. In the case of specimens incubated in Ami, the lowest R was noted at the level of 3101 Ω × cm². There were statistically significant changes in R between the control and study groups incubated in Ami and Bume solutions, respectively (

Table 2. Results of the Mann–Whitney test for transepithelial potential (PD, mV) and transepithelial resistance (R, Ω × cm²) measured under stationary conditions for the control skin specimens treated with the iso-osmotic Ringer solution (RS) and specimens frozen with cryoprotection and incubated in RS, amiloride (Ami, 0.1 mmol/L), or bumetanide (Bume, 0.1 mmol/L) solutions.

Parameter	Control vs. RS (p)	Control vs. Ami (p)	Control vs. Bume (p)	RS vs. Ami (p)	RS vs. Bume (p)	Ami vs. Bume (p)
R	0.0487	<0.001	0.1632	<0.001	0.0055	0.0107
PD	<0.001	0.8331	0.2122	0.01	0.0094	0.3433

Abbreviations: Control—specimens incubated in RS; p < 0.05 is considered statistically significant.

).

Measurement of transepithelial PD under stationary conditions revealed the presence of transepithelial ion transport. The median value for the control group was −0.27 mV. In the study group, the skin fragments demonstrated a constant electrical potential with a median of 0 mV for both RS and Ami incubations, whereas for the Bume incubation, the median PD value was −0.12 mV (

Table 3. Values of minimal (PDmin, mV) and maximal (PDmax, mV) transepithelial electric potential measured during 15-s series of mechanical and mechanical–chemical stimulations for the control skin specimens and samples frozen with cryoprotection and incubated in the iso-osmotic Ringer solution (RS), amiloride (Ami, 0.1 mmol/L), or bumetanide (Bume; 0.1 mmol/L) solutions.

		Incubation
		Control (n = 23)		RS (n = 21)		Ami (n = 26)		Bume (n = 26)
Stimulation	Parameters	PDmin (mV)	PDmax (mV)	PDmin (mV)	PDmax (mV)	PDmin (mV)	PDmax (mV)	PDmin (mV)	PDmax (mV)
RS	median	−0.46	0.4	−0.92	0.64	n/o		n/o
	upper quartile	−0.27	1.34	−0.27	1.65
	lower quartile	−1.07	0	−1.78	0.27
Ami	median	−0.37	0.34	−0.76	0.64	−0.34	0.58	−0.46	0.29
	upper quartile	−0.12	1.8	−0.21	0.85	0.49	2.14	−0.34	0
	lower quartile	−1.28	−0.36	−1.59	0.43	−0.92	0	−0.89	0.64
Bume	median	−0.49	0.15	−0.70	1.01	−0.38	0.19	−0.34	0.34
	upper quartile	0	1.16	−0.15	1.53	0	0.61	−0.18	1.07
	lower quartile	−1.25	−0.44	−1.65	0.34	−1.22	−0.11	−0.55	0.15
AB	median	−0.19	0.21	−0.76	1.01	−0.37	0.21	−0.44	0.41
	upper quartile	0.82	0.98	−0.27	1.46	0	0.76	−0.21	1.71
	lower quartile	−0.95	−0.34	−1.83	0.24	−1.68	−0.12	−0.89	0.18

Abbreviations: n—number of the skin specimens; AB—solution of amiloride (0.1 mmol/L) and bumetanide (0.1 mmol/L).

).

The PDmin and PDmax values measured for both control and study skin specimens incubated in RS were lower than those measured during mechanical–chemical stimulation. PDmin and PDmax were measured after mechanical and mechanical–chemical stimulations of the samples frozen with cryoprotection and were modified compared to those of the non-frozen tissue samples that were stimulated similarly (Table 3).

The electric potential measured under stationary conditions (PD) in each case significantly differed from that measured during stimulations (PD vs. PDmin and PD vs. PDmax). Stimulations triggered repeatable changes in ion transport regardless of the freezing procedure used. However, those changes depended on the type of stimulation (mechanical or mechanical–chemical) used (

Table 4. Results of the Wilcoxon test (p < 0.05) for transepithelial electric potential measured under stationary conditions (PD, mV) and during 15-s stimulations (minimal PD, PDmin, and maximal PD, PDmax, mV) for the control skin specimens and the samples frozen with cryoprotection and incubated in the iso-osmotic Ringer solution (RS), amiloride (Ami, 0.1 mmol/L), or bumetanide (Bume, 0.1 mmol/L) solutions.

Stimulation (15 s)	Control (p)	RS Incubation (p)	Ami Incubation (p)	Bume Incubation (p)
Bume
PD vs. PDmax	<0.001	<0.001	0.0014	<0.001
PD vs. PDmin	<0.001	<0.001	<0.001	<0.001
PDmax vs. PDmin	<0.001	<0.001	<0.001	<0.001
Ami
PD vs. PDmax	<0.001	<0.001	<0.001	<0.001
PD vs. PDmin	<0.001	<0.001	<0.001	0.0199
PDmax vs. PDmin	<0.001	<0.001	0.001	<0.001
AB
PD vs. PDmax	<0.001	<0.001	<0.001	<0.001
PD vs. PDmin	<0.001	<0.001	<0.001	0.0028
PDmax vs. PDmin	<0.001	<0.001	<0.001	<0.001

Abbreviations: AB—solution of amiloride (0.1 mmol/L) and bumetanide (0.1 mmol/L); p < 0.05 is considered statistically significant.

).

The use of mechanical stimulation (RS solution) caused statistically significant changes in the measured PDmin and PDmax values in both frozen and non-frozen tissue specimens (

Table 5. Results of the Mann–Whitney test for transepithelial potential measured during 15-s series of stimulations (minimal PD, PDmin, and maximal PD, PDmax, mV) for the control skin specimens and the specimens frozen with cryoprotection and incubated in the iso-osmotic Ringer solution (RS), amiloride (Ami, 0.1 mmol/L), or bumetanide (Bume, 0.1 mmol/L) solutions.

Stimulation	Control vs. RS (p)	Control vs. Ami (p)	Control vs. Bume (p)	RS vs. Ami (p)	RS vs. Bume (p)	Ami vs. Bume (p)
Bume
PDmin	0.3717	0.1393	0.0404	0.7898	0.2136	0.8388
PDmax	0.0539	0.0031	0.1455	0.7332	0.1518	0.2635
Ami
PDmin	0.1451	0.1902	0.2221	0.7484	0.4828	0.6666
PDmax	0.3592	0.0005	0.0653	0.2924	0.7635	0.0952
AB
PDmin	0.1729	0.4931	0.3351	0.5815	0.9542	0.8474
PDmax	0.1551	0.0888	0.6631	0.7713	0.1302	0.1841

Abbreviations: AB—solution of amiloride (0.1 mmol/L) and bumetanide (0.1 mmol/L); p < 0.05 is considered statistically significant.

). Comparing both groups revealed statistically different PDmin and PDmax values measured during the stimulation with RS. The use of blockers of the transepithelial routes of sodium (Ami) and chloride (Bume) ion transport, also in combination with (AB), exhibited statistically significant differences in their responses, similar to those observed after the stimulation with RS. The responses of the frozen skin fragments were more intense in the case of both the mechanical (RS) and the mechanical–chemical (Ami, Bume, AB) stimuli. The most significant differences were observed in the case of the chloride ion transport phase response to the solution Ami.

4. Discussion

Storing tissue specimens in conditions ensuring their unaltered state and preserving the physiology of their biochemical processes is an essential requirement. It facilitates transplant acceptance and stimulation of the patient’s tissue to initiate regeneration and healing processes, thus enabling therapeutic success [2,3,4,5,8]. The presented study evaluated ion transport changes in skin fragments stored by freezing at −80 °C. The examined tissue samples were perfused with a DMSO solution to prevent damage to the internal cell structure by frozen water crystals [2,3,4,28,29]. We used a procedure of rapid freezing to −80 °C and slow thawing, which corresponded to procedures used for skin fragments for transplantation by organ banks [1,3,5,13].

The measured resistance values did not show statistically significant differences, comparing the control fragment with the frozen ones, except for the incubation in Ami (Table 2). Kasting and Bowman [18] and de Silva Serra et al. [30] obtained similar results for frozen and non-frozen skin samples. This indicates that the experimental procedures did not cause any measurable damage to the tissue structure or any changes in the adhesion of cells in the examined specimens [30]. The constant electrical resistance indicates that there was no deformation due to changes in cell hydration or ion permeability in both the extracellular space and the cell interior [16,31,32,33,34,35]. The examined skin fragments were characterized by a compact, live cell structure capable of transporting ions [4,28,31]. It could be assumed that the microspaces that maintain the appropriate skin texture in the frozen tissue fragments [13] were preserved, i.e., quantitatively, they remained at the physiological level. The observed decrease in resistance of 30% for tissues subjected to freezing and incubated in RS and 50% for incubation in Ami for the control could be explained by the change in the permeability of frozen tissues to ions and water, and not by extensive damage to cells and/or intercellular spaces. Despite significant changes in the resistance of frozen tissues, they were similar to the measurements obtained on non-frozen fragments treated with diclofenac [36] or vitamin A [37].

It has been proven that assessing viability and preserving the mechanisms of transepithelial ion transport in tissues intended for transplantation are extremely important for predicting pain reactions, hypersensitivity, and transplant rejection [24]. In the present study, the frozen skin fragments were more reactive than the non-frozen fragments, as evidenced by the differences between the PDmax and PDmin values measured during mechanical stimulation using RS (Table 4 and Table 5). Likewise, the use of mechanical–chemical stimulation increased sodium ion transport in response to bumetanide, and chloride ion transport in response to amiloride. Changes in sodium ion transport trigger water influx into cells, which can be important when thawing skin fragments subjected to dehydration using DMSO before freezing [4,16,27,28]. Interestingly, the freezing procedure employing DMSO caused repeatable changes in the transport of sodium ions (Table 3 and Table 4). Thus, the movement of water between cells and the extracellular space seems to be induced by thawing [2,16,29], and using a cryoprotectant makes the process less intense and/or slower [1,4]. Water transport leads to equalization of the concentrations of small molecular substances [4,8,29,38,39], including ions, and the use of DMSO in the freezing procedure could impact the intensification of that transport [4]. Similarly, increased transport of sodium ions was observed after contact with a mechanical stimulus in experiments with the biopesticide deltamethrin, which may be the cause of undesirable skin reactions when working with this compound [27].

The release of sodium ions by keratinocytes into the extracellular space can cause hyperactivity and release neurotransmitters from free nerve terminals, which in turn can lead to the stimulation of C-fibers and increased nociception [15,24]. Therefore, increased absorption of sodium ions from the extracellular space should protect against the stimulation of C-fibers and excessive pain response. However, an increased activity of ENaC and a more intense sodium ion transport by keratinocytes were proven to be responsible for the development of hypersensitivity and/or allergic reactions [36,40]. Thus, intense ion transport by the sodium channels located on keratinocytes [19] can be associated with the hyperreactivity of tissue fragments that have been frozen before transplantation [24,40]. Furthermore, retaining the proper activity of sodium channels is associated with the onset of regeneration processes in keratinocytes and fibroblasts [18,19,20,21]. After the transplantation of fragments of previously frozen skin, this phenomenon may lead to benefits such as accelerated healing and regeneration.

The frozen skin fragments were characterized by an increased release of chloride ions during stimulation compared to the transport of sodium ions (Table 3, Table 4 and Table 5). The intensity of the chloride ions transport changes, measured as PDmax and PDmin, seems to be important in combating the abrupt changes in water flow and, indirectly, in protecting skin fragments against the initiation of inflammatory response [20,25,38]. After a sudden influx of water, frozen keratinocytes can release proteins, including pro-inflammatory factors, into the extracellular space [8,39], leading to the migration of immunocompetent cells into the area with altered ion composition. However, the release of chloride ions causes water efflux from the cells into the intercellular space, leading to the dilution of the extracellular fluid and substances dissolved therein [25]. Furthermore, changes in hydration can induce differences in the transport of therapeutics into the skin tissue, particularly those administered into the skin area [8,16,36,37]. Intensification of the transport of chloride and sodium ions in frozen skin fragments can contribute to changes in the gradient of active substances and their improper diffusion within the skin [8,39,40].

In the presented study, blocking the sodium ion transport made the transport of ions less intense. The applied inhibitor, amiloride, is characterized by reversible and non-competitive inhibition [16,17,36,37]. Additionally, it is easy to rinse it from the tissue samples. Therefore, treating frozen-and-thawed tissue specimens with a low concentration of amiloride solution might help to return the transport mechanisms to the pre-freeze state. It seems that, quantitatively, the microspaces were not modified during the freezing and thawing procedure, but their osmolality changed, which may additionally modify ion transport, especially the accumulation of sodium ions. The storage of sodium ions in the skin is, to some extent, a physiological phenomenon [12,37,38,40]. However, the rapid, intense influx of sodium into keratinocytes after tissue thawing changes the environment around the cells and may affect their reactivity and functions [40]. It appears that freezing skin fragments with DMSO as a cryoprotectant significantly alters the activity of ion channels rather than the sodium-potassium pump and the sodium-chloride-potassium transporter. The continuous PD measurement reflects the constantly occurring ion transport, which was higher during the frozen tissues’ incubation in Ami.

The blockage of the chloride ion transport path also reduced the response to mechanical–chemical stimuli, in a manner similar to that of the fragments that were not subjected to the freezing procedure. However, the range of potential changes measured during stimulation was significantly higher (p = 0.0404). It appears that the use of chloride ion transport blockers is of less importance for maintaining the ion transport at an optimal level. However, the incubation duration in bumetanide solution in the presented experiment might be too short to restore homeostasis in the studied skin fragments.

The above-described experiment on freezing procedure and using DMSO as a cryoprotectant for skin fragments demonstrated differences in the transport of sodium and chloride ions, which can be associated with therapeutic difficulties in patients requiring treatment with the use of skin fragments and their substitutes.

5. Conclusions

The results show that the skin subjected to freezing in DMSO solution is alive, compact, and durable. However, the examined samples presented unphysiologically increased transport of sodium and chloride ions, which may translate into a change in the perception of pain, the development of hypersensitivity and/or allergy reactions, and the initiation of repair and regeneration processes. Therefore, using the cryoprotectant was ineffective in protecting the frozen skin samples from modifications in ion transport. Additionally, using a sodium ion transport blocker during the thawing procedure could be considered, but this requires further experiments. It can be concluded that there is a constant need for new methods to maintain the proper course of physiological processes, including ion transport, in frozen skin fragments intended for transplantation.