Vibration Technology Makes It Possible to Obtain Standardized Biological Preparations: Vibrational Iterations Based on Cultured Cells

Abstract

Cell-based therapy is a promising direction for the treatment of various diseases. However, it is associated with several problems, primarily related to reproducibility and standardization. In this context, the development of new methods for the production of cell-based preparations is of particular relevance. Recently, a novel technology named ‘crossing’ has been developed. It comprises the multi-stage vibrational processing of two closely spaced test tubes containing the initial substance and a neutral carrier (water or lactose). As a result, the neutral carrier acquires some properties of the initial substance, and artificial products, vibrational iterations, are obtained. Some vibrational iterations are also capable of exerting a modifying effect on the initial substance (or its target in the body), changing its physico-chemical/biological properties. Earlier, we demonstrated the possibility of obtaining vibrational iterations from biological molecules (antibodies). In this study, we evaluated the biological effects of vibrational iterations obtained by the crossing technology using cells grown in culture. This work shows that vibrational iterations obtained from CHO-S cell culture affect the ability of CHO-S cells to utilize glucose in the presence of insulin. The data demonstrate the prospect of developing fundamentally new biological drugs based on vibrational iterations, including for the treatment of diabetes mellitus.

1. Introduction

Every year, the niche of cell-based therapeutics in the pharmaceutical market is becoming more and more extensive [1,2]. However, the development of cell-based drugs using conventional biotechnology approaches faces many challenges. One of the main problems is the reproducibility of the results, since cell-based drugs can exhibit significant variability in their properties and efficacy [3]. This variability is due to differences in cell sources, culture conditions, and processing methods, which complicates the standardization and mass production of such preparations. The lack of standardized methods for assessing the quality and efficacy of cell preparations hinders their clinical application and regulatory approval [4]. Standardization includes not only control over the manufacturing process, but also the development of reliable methods for testing the biological properties of cells, such as their viability, differentiation potential, and ability to interact with other cells and tissues [3].

Recently, a technology for the preparation of vibrational iterations, crossing, was developed [5]. Two closely spaced vials containing the initial substance and a neutral carrier (water or lactose) are subjected to vibration treatment. As a result, the neutral carrier acquires some properties of the initial substance. Thus, an artificial product, vibrational iteration, is obtained. Some vibrational iterations are also capable of affecting the initial substance or their target, changing its physico-chemical or biological properties [5,6], which expands the possibilities of using vibrational iterations for therapeutic purposes.

During our study, we found that vibrational iterations with different physico-chemical properties differ in the spectrum of biological activity. This feature allows us, firstly, to select the most suitable fractions of vibrational iterations for a specific task, and secondly, simplifies the validation of biological preparations based on vibrational iterations by analyzing their physico-chemical properties. It has been demonstrated that samples prepared using vibration technology exhibit changes in the hydrogen bond network, which can be investigated in detail using terahertz (THz) spectroscopy and conductometry [7,8,9]. In turn, microwave radiometry (in the gigahertz (GHz) range) can reveal a dramatic increase in the number of nanobubbles during shaking [10]. Thus, these three methods were selected for characterization of vibrational iterations.

Previously, we studied vibrational iterations obtained using various initial substances, including biological molecules (antibodies). In the current study, we aimed to assess the effects of vibrational iterations obtained using live Chinese hamster ovary (CHO) S cells grown in culture. CHO-S is a subline of the original CHO cell line that grows in suspension culture. CHO-S cells do not require serum supplementation and are widely used to produce biologics in the biopharmaceutical industry. Insulin is the only hormone required for the continued growth of CHO-S cells in the defined medium. The dynamics of glucose uptake and the kinetics of cell product release are usually studied using these cells [11,12].

Recently, it has also been shown that vibrational iterations prepared using glucose powder as the initial substance can affect glucose consumption by CHO-S cells [5]. We suggest that this model can also be used to study vibrational iterations obtained from CHO-S cells grown in culture. These iterations were added to fresh CHO-S culture, and glucose consumption was measured in the presence of different insulin concentrations.

2. Materials and Methods

2.1. Sample Preparation

All vibrational iterations for each study were prepared by the same operator on the same day using borosilicate glass vials (60 mL, G075G-27/140-H Glastechnik, Grafenroda, Germany) or flasks (500 mL, Simax, Czech Republic). During sample preparation and subsequent measurements, the laboratory temperature was monitored using an IVA-6N thermohygrometer (NPK MICROFOR LLC, Moscow, Russia), maintaining it at 22 ± 1 °C.

Type 1 ultrapure water with a resistivity of 18.2 MOhm × cm (Milli-Q Integral 5, Millipore, Molsheim, France), hereafter referred to as ‘purified water’, served as the neutral carrier and was also used for all other purposes. The water’s quality was checked daily in terms of resistivity (by a SevenCompact S230 conductometer (Mettler Toledo, Greifensee, Switzerland)) and pH (by a SevenCompact S220 pH meter (Mettler Toledo, Greifensee, Switzerland)).

For handling liquids, automatic variable volume pipettes (Eppendorf, Hamburg, Germany; Socorex, Greifensee, Switzerland) along with accuracy class A measuring glassware (Borosil, Mumbai, India; Steklopribor, Zavodske, Ukraine) were employed. Vibration treatments were conducted using an MS 3 basic shaker (IKA-Werke, Staufen, Germany) equipped with a standard insert (frequency: 3000 rpm, time: 10 s, and diameter of the shaker orbit: 4.5 mm).

The process of obtaining iterations (crossing) took place in several stages (Figure 1). Samples were prepared according to the technology described in [5], except that CHO-S suspension was used as the initial substance. CHO-S cells (R80007, Thermo Scientific, Carlsbad, CA, USA) were used at the 20th passage. The culture was maintained in Hybris-1 serum-free culture medium (C740p, PanEco, Moscow, Russia) with L-glutamine (PanEco, Moscow, Russia, F032) and penicillin–streptomycin (A065, PanEco, Moscow, Russia). To prepare the cell suspension (initial substance), 5 mL of 72 h CHO-S cell culture (1 × 10⁵ cells/mL with 95% viability) was added to a 60 mL vial filled with 45 mL of culture medium and gently mixed without using a vortex. Next, the vial with the initial substance was placed close to a 60 mL vial filled with 50 mL of intact purified water (neutral carrier), and both vials were vortexed for 10 s, followed by incubation at room temperature on the bench top for 1 min. After this procedure, the vial containing purified water was considered as iteration zero of CHO-S cell culture. Next, vibrational iteration No. 1 was obtained by placing the vial with vibrational iteration zero close to a 500 mL flask filled with 480 mL of intact purified water and vortexed as in the previous step. After this procedure, the vial containing purified water was considered as iteration No. 1 of CHO-S cell culture. The procedure was repeated until we obtained vibrational iteration No. 7. A calibrated laboratory timer (Traceable, VWR, Darmstadt, Germany) was used to monitor incubation times.

2.2. Analysis of Iterations

General procedures for performing conductometry, radiometry, and THz spectroscopy for classification of vibrational iterations into fractions have been described in detail previously [5]. On the day of analysis, vibrational iterations were vortexed once for 10 s at 3000 rpm. Each sample was analyzed using conductometry (3 aliquots were measured in 3 repetitions), radiometry (a single aliquot was measured in 5 or 9 repetitions), and THz spectroscopy (5 aliquots were analyzed in 2 repetitions).

2.2.1. Conductometry

Conductivity measurements were performed using a SevenCompact S230 conductivity meter with a two-pole conductivity cell having a constant 0.1 cm⁻¹ in the range from 0.001 μS/cm to 500 μS/cm. Measurements were performed using a non-linear temperature correction mode for temperature compensation. The measurement procedure began by immersing the probe in the specimen without contact with the walls or the bottom of the tube to avoid interference. The readings were allowed to stabilize before recording; this varied depending on the sample, but usually required up to 20 s. Before each measurement, the electrode was washed with purified water, after which the electrical conductivity was measured using a new aliquot of the sample. Between samples, the probe was thoroughly washed with purified water and dried with a clean, lint-free laboratory wipe to prevent cross-contamination.

2.2.2. Radiometry

In order to prevent external electromagnetic interference, radiometry measurements were conducted inside an in-house-build Faraday cage made of an aluminum frame and copper mesh. A TES-92 electromagnetic radiation detector (TES Electrical Electronic Corp., Taipei, Taiwan) was used to measure the radiation flux density, which was produced by the samples. During measurement, the following parameters were set: energy flux density measurement range (0 μW/m²–30.93 W/m² with a resolution of 0.001 μW/m²); measurement method (isotropic); sensor (three-channel); frequency range (50 MHz–3.5 GHz); display refresh rate (about 400 ms); units (μW/m²); and measurement mode (MAX AVG). An ADF4351 signal generator (Diymore, Shenzhen, China) with a 1.4–10.5 GHz UWB directional high-gain antenna (ZWAAJKQK, Shenzhen, China) was used as an external radiation source. Inside the cage, devices were attached to a clamp stand or a specially designed metal frame so that the distance from the detector sensor to the sample was 0.5 cm. To control the temperature, a WF-4000 infrared laser thermometer (B.Well Swiss AG, Widnau, Switzerland) was used.

The measurement procedure was as follows: a 10 mL sample was placed in a polystyrene single-use Petri dish with a diameter of 60 mm (Perint, Saint Petersburg, Russia) using a manual macropipette (Acura 835 1–10 mL, Socorex, Ecublens, Switzerland) and closed with a lid. The thermal shaker (Biosan, Riga, Latvia) for preheating was heated to 52 °C, and the sample was heated to a temperature of 37 °C after closing the lid. The heating time was 4–5 min, and the temperature was monitored with an infrared laser thermometer. The required measurement parameters of the TES-92 device were set at measurement axis—Z, units of measurement—μW/m². After that, the sample was placed on a thermal shaker (or hot plate) inside the cage, where the temperature was set to 45 °C to maintain the desired temperature of the sample, and the Petri dish was placed in the center of the heating element for more uniform heating. The antenna was aimed at the sample, and the radiation parameters were selected: the frequency was set to 2200 MHz and power was set to +5 dBm. The Faraday cage was then closed, and measurements were performed in MAX AVG mode, selecting this mode using the MAX AVG button placed outside the cage. The measurement time of one replicate was 10 min and was controlled by a laboratory timer. After the specified time, the radiation flux density value from the TES-92 display was recorded, the Faraday cage door was opened, the sample was removed, and another sample was put in its place and preheated to the desired temperature, after which subsequent measurements were conducted.

2.2.3. Terahertz Spectroscopy

For THz spectroscopy, 9 mL of tissue culture supernatant (conditioned medium of the 20th passage CHO-S cells) was collected. Then the supernatant was passed through a PVDF membrane syringe filter with a pore size of 0.2 μm (729219, Macherey-Nagel, Düren, Germany) and stored in a refrigerator (2–8 °C). On the day of the experiment, a 10-fold diluted aqueous solution of the conditioned CHO-S medium was freshly prepared. Each solution was prepared in three independent replicates, for each of which two independent measurements were performed. All samples were analyzed randomly.

To assess the effect of vibrational iterations on the conditioned CHO-S medium, a mixture of the diluted cell culture medium and the vibrational iteration samples was prepared in a volume of 1 mL. For this purpose, 990 μL of the conditioned CHO-S medium (preliminarily diluted 10 times) and 10 μL of the vibrational iteration sample (obtained as described in Section 2.1) was added to an Eppendorf tube. The resulting solution was mixed by gently inverting the tube five times.

Sample spectra were recorded on a TPS Spectra 3000 THz spectrometer (Teraview Ltd., Cambridge, UK) in the range of 0.1–275 cm⁻¹ with a spectral resolution of 4 cm⁻¹. The Fourier transform of the measured time functions of electric field strength into spectra was performed using the Blackmann–Harris 3 apodization function. To obtain one spectrum, 1200 scans were averaged. The temperature of all solutions during the measurement was maintained at 25 ± 0.5 °C by placing them in a cuvette in a 4000 Series High-Stability Temperature Controller thermostatic holder (Specac Inc., Fort Washington, PA, USA). Samples were analyzed in two identical quartz cuvettes, differing only in inter-window distance (approximately 50 and 100 μm thick). The exact distances between the windows were determined interferometrically after the cuvettes had been assembled. For this purpose, the transmission spectra of the empty cuvettes were measured on a FT-801 (OOO “NPF “Simex”, Novosibirsk, Russia) in the range of 4000–8000 cm⁻¹.

The single-beam radiation intensity spectrum of a sample aliquot recorded in a 100 μm cuvette was divided by a similar spectrum of another aliquot of the same sample recorded in a 50 μm cuvette to obtain a transmission spectrum of this sample with a thickness equal to the difference in the thicknesses of the two cuvettes. The phase spectrum of the sample in a smaller thickness cuvette was subtracted from the phase spectrum of the sample in a larger thickness cuvette, which, after dividing by the difference in thickness of the two cuvettes, gave the refractive index spectrum of the sample.

Transmittance and refractive index spectra were used to calculate the spectra of the real and imaginary components of the dielectric constant of the solutions. The latter were used to calculate the coefficients de1 (∆ε1) and de2 (∆ε2), which depend on the binding of water molecules in the test solution. Thus, the increased binding of water molecules in hydrate shells is accompanied by a decrease in the amplitude of the Debye relaxation band (de1). At the same time, it is possible to both increase and decrease the amplitude of the high-frequency relaxation band (de2) in different cases. The calculation of de1 and de2 values was carried out in Python v.3.8.10 in the narrowed frequency range from 10 cm⁻¹ to 110 cm⁻¹. The dried clean cuvette was closed, and the housing was dried from the outside with a pressurized nitrogen jet and placed in the THz cuvette compartment of the spectrometer. A pause of 5 min was maintained to stabilize the temperature and purge the optical part of the spectrometer with dried air.

2.3. Classification of Vibrational Iterations into Fractions

The obtained samples can be classified into 4 groups (fractions) depending on the results of the analysis of their properties [5]:

• Native—iterations which, according to the results of conductometry and radiometry, do not have significant changes in physico-chemical properties compared with an intact neutral carrier (water) and, according to the results of THz spectroscopy, do not have a modifying effect.
• Semi-Native—iterations in which statistically significant changes in physico-chemical properties were found compared with an intact neutral carrier (water), but which, like ‘Native’ iterations, do not have a modifying effect based on THz spectroscopy data.
• Semi-Active—iterations that do not have significant changes in physico-chemical properties compared with an intact neutral carrier (water), but at the same time, unlike ‘Native’ iterations, have a modifying effect according to THz spectroscopy.
• Active—iterations that have significant changes in physico-chemical properties compared to an intact neutral carrier (water) and at the same time have a modifying effect according to THz spectroscopy.

The test result was considered positive (+) if it met the acceptance criteria. Otherwise, the result was taken as negative (−) (Figure 1). The acceptance criteria are as follows: the values obtained for vibrational iterations should statistically significantly (p < 0.05) differ from those of intact neutral carrier (water) by ±5% or more (by conductometry and THz spectroscopy) or by +10% or more (by radiometry). The values obtained for intact neutral carrier (water) were taken as 100%.

2.4. Estimation of the Amount of Glucose Consumed by CHO-S Cells Depending on Insulin Concentration

2.4.1. Vibrational Iteration Samples

In order to assess the impact of vibrational iterations on glucose uptake by CHO-S cells, the I5 (‘Semi-Native’) and I7 (‘Active’) fractions were utilized. These fractions were selected based on the results of conductometry, radiometry, and THz spectrometry, as described in Section 2.2 and Section 2.3.

2.4.2. Cell Line

The sample activity was evaluated using the same cell line (CHO-S) that was used to prepare vibrational iterations. General procedures have been described in detail [5]. Briefly, CHO-S cells were grown in 12-well plates (3512, Corning, Glendale, AZ, USA) in Hybris medium without insulin (PanEco, Moscow, Russia) over a period of 7 days under standard conditions (37 °C, 5% CO₂). Subsequently, the cells were seeded into 96-well plates (3599, Corning, Glendale, AZ, USA), followed by the addition of test samples and human recombinant insulin solution (PanEco, Moscow, Russia) at varying concentrations (0.5 μg/mL, 5 μg/mL, and 50 μg/mL). The samples were filtered through a 0.2 µm pore-sized PVDF membrane syringe filter (729219, Macherey-Nagel, Düren, Germany) before being introduced into the cell culture at a 1:10 dilution.

The final cell concentration was adjusted to 0.15 × 10⁶ cells/mL. After incubation for 72 h at 37 °C with 5% CO₂, glucose consumption analysis was conducted for each experimental well using the hexokinase method, and WST assay was used to determine the normalized glucose consumption levels.

2.4.3. Hexokinase Method

In 2 mL plastic tubes (CFT001020, JetBiofil, Guangzhou, China), containing 780 µL of 0.1 M Tris-HCl buffer, pH 7.8 (Sigma-Aldrich, Saint Louis, MO, USA), 20 µL of cell suspension taken from the respective well with the sample was added. The mixtures were vortexed (Biosan, Riga, Latvia), and 50 μL aliquots were transferred into the wells of a 96-well plate (Corning, Glendale, AZ, USA).

Subsequently, 50 µL of an enzyme and coenzyme mixture (comprising Tris buffer, hexokinase, NAD+, and ATP) was added to the wells, and plates were incubated for 1 h at 37 °C. Following this incubation step, 200 µL of Tris buffer (0.1 M, pH 7.8) was dispensed into each well. The optical densities were recorded by a Multiskan FC microplate reader (Thermo Scientific, Waltham, MA, USA) at wavelengths of 340 nm and 450 nm. Corrected optical density values for each well were determined using the following formula: OD_glu = OD₃₄₀ − OD₄₅₀ − OD_{tris(calibration).} Thereafter, glucose consumption was computed using the formula C_glu = 30 − ((OD_glu − b)/a × 40), based on the calibration equation y = ax + b. For replicates within a given insulin concentration, the mean, standard deviation, and coefficient of variation were calculated using Excel 2007 software (Microsoft, Redmond, WA, USA).

2.4.4. WST-Assay

To assess cellular metabolic activity, the WST-1 reagent (FI28731, Biosynth, Suzhou, China) was employed. The assay was carried out following the manufacturer’s protocol. CHO-S cells were seeded in duplicate into 96-well plates (3599, Corning, Glendale, CA, USA) at 100 μL per well, followed by the addition of 8 μL of WST-1 reagent per well. The plates were then incubated for 3 h at 37 °C. Optical density measurements were taken on a Multiskan FC microplate reader at wavelengths of 440 nm and 650 nm. The corrected optical density was calculated by the following formula: OD_wst = OD₄₄₀ − OD₆₅₀. The calculation of normalized glucose uptake by CHO-S cells was carried out in accordance with the following formula: N = C_glu/OD_wst (where OD_wst is the average value of two replicates).

The test result was considered positive if it met the acceptance criteria: the values obtained for vibrational iterations should statistically significantly (p < 0.05) differ from those of intact water by 15%.

2.5. Statistical Analysis

Outliers in groups were determined using the interquartile range. Groups were compared using Student/Welch t-test and Kruskal–Wallis test, followed by Dunn’s post hoc test. Groups were compared for glucose consumption at each insulin concentration point, and p-values were corrected using Holm’s method. Differences between groups were considered statistically significant at p < 0.05.

3. Results and Discussion

3.1. Characteristics of Fractions of Vibrational Iterations

Using crossing, a series of vibrational iterations was obtained. According to their physical characteristics (Supplementary Information), vibrational iterations from No. 1 to No. 7 were classified into three groups (fractions): Semi-Native, Semi-Active, and Active. Vibrational iterations No. 5 and No. 7 were selected for further studies. The summary of physico-chemical characteristics for selected fractions is presented in

Table 1. Physico-chemical properties and modifying effect of vibrational iterations.

Sample	Conductometry	Radiometry	THz Spectroscopy (de1)	THz Spectroscopy (de2)	Fraction
Vibrational iteration No. 5	−2%	17% *	1%	3%	Semi-Native
Vibrational iteration No. 7	−12% *	5%	0%	7% *	Active

Note: * Meets the acceptance criteria (p < 0.05 according to conductometry and THz spectroscopy (difference more than ±5%) and p < 0.05 according to radiometry (difference more than ±10%)) compared to intact neutral carrier (water) (see Section 2.3). Data are normalized to the corresponding values obtained for intact neutral carrier (water).

.

Vibrational iteration No. 5 appears to belong to the ‘Semi-Native’ fraction: (1) the conductometry results show a significant change in its physico-chemical properties compared to the intact neutral carrier (water) (SEC: 2%, p < 0.0001; EMR flux density: 17%, p < 10⁻⁶); (2) THz spectroscopy results show that vibrational iteration No. 5 did not change the characteristics of the tissue culture supernatant of the original cells (del: 1% vs. water, p = 0.23 and de2: 3% vs. water, p = 0.29). Vibrational iteration No. 7 appears to belong to the ‘Active’ fraction: (1) statistically significant differences in physical properties were revealed compared to the intact neutral carrier (water) according to conductometry (12%, p < 10⁻²⁰); (2) it has a modifying effect on the dielectric properties of the tissue culture supernatant of the original CHO-S cells as determined by THz spectroscopy (7% vs. water, p < 0.05 for de2). Specifically, a change in the value of the coefficient de2 (i.e., the amplitude of the high-frequency relaxation band) reflects an alteration in the amount of bound water molecules in the studied solution.

3.2. Biological Activity of Vibrational Iterations

To assess biological activity, vibrational iterations No. 5 and No. 7 were tested in an in vitro cell culture model. After 72 h incubation, their effects on glucose consumption by CHO-S cells were analyzed (Figure 2).

In two independent experiments it was shown that, relative to a neutral carrier (water), vibrational iteration No. 7 (‘Active’) statistically significantly reduced and vibrational iteration No. 5 (‘Semi-Native’) statistically significantly increased the consumption of glucose by CHO-S cells in the presence of various concentrations of insulin. Thus, vibrational iterations with different physico-chemical properties have different biological effects.

The ability of post-vibrational transfer of part of the biological properties of an initial substance, which in this case was a cell culture, to a neutral carrier is a recently discovered phenomenon that requires an explanation from theoretical physics. In fact, this is an interaction without direct contact between an initial substance and a neutral carrier (non-contact or distant effect). We believe that this topic will be a subject for future research. However, this is not a unique process with no natural analogs. In the organism, there are numerous examples of interactions between cells that occur at a distance and without the use of chemical mediators, for example, through sound, electric current and electromagnetic radiation [13,14].

By now, it is hypothesized that at least two factors contribute to the long-term post-vibrational effects of vibrational iterations: radiative and non-radiative components, as in the case of distant effect of high dilutions. The radiative component of the distant effect of high dilutions depends quadratically on the distance from the acceptor and is directly proportional to the duration of joint incubation, as described in [15,16]. This resembles the model of a photochemical reaction. Additionally, the non-radiative component has been confirmed by the relaxation of the highly diluted solution’s (donor) properties after remote exposure to the acceptor. This can be explained by a model similar to inductive resonance energy transfer, as proposed in [17]. The need for vibration treatment to manifest the characteristic GHz radiation of such samples, as well as their distant effect on the acceptor, is described in the model of nanobubble oscillations (shape deviations from the equilibrium spherical state) in an aqueous solution [10]. The number of nanobubbles increases sharply after vibration treatment. When nanobubbles move in a convection flow of liquid, it leads to the displacement of ions stabilizing nanobubbles, and oscillation of nanobubbles occurs. The GHz radiation of nanobubbles is determined by their oscillation with frequency in the GHz range. Previous studies have confirmed that the preparation of samples without mechanical action leads to the absence of their activity [18]. CHO-S cells here are a model that allows us to detect and observe these effects.

Therefore, the crossing technology opens up new prospects for therapy with drugs obtained from biological substances. The biological activity of vibrational iterations allows us to move from the use of live cells to their technologically obtained ‘replicas’, which are easy to standardize by physico-chemical, primarily spectral characteristics and reproducible biological action. Thus, we consider our results as an opportunity to move from cell-based, individually applied technologies, to the mass use of biological drugs of a new type.