Research
Biotechnol Ind J, Volume: 15( 2)

Development and Validation Method for the Determination of Sildenafil Citrate Tablets by using UV-Spectrophotometer in Pharmaceutical Formulation

*Correspondence:
João Tadeu Ribeiro-Paes , Laboratory of Genetics and Cell Therapy GenTe Cel, Department of Biotechnology São Paulo State University, São Paulo, Brazil, E-Mail: [email protected]

Received: November 28, 2018; Accepted: December 07, 2018; Published: December 15, 2018

Citation: Fuoco N, Oliveira RG, Marcelino MY, et al. Efficient Isolation and Proliferation of Human Adipose-Derived Mesenchymal Stromal Cells in Xeno-Free Conditions. Biotechnol Ind J. 2018;15(2):181.

Abstract

The objective of this paper is to describe the optimization, validation, and application of spectrophotometric technique for determination of Sildenafil Citrate in their pharmaceutical formulation (tablets). In this paper, a simple, rapid, accurate and sensitive spectrophotometric method has been developed and validated. The method is a direct spectrophotometric method based on thawed of sildenafil citrate in diluted hydrochloric acid. The maximum absorption wavelength for the determination of Sildenafil citrate was found to be 295 nm. Under the optimized condition, Beer’s law was obeyed in the concentration range from 5.0-40.0 μg/ml.

Keywords

Mesenchymal stromal cells; Stem cells; Adipose tissue; Fetal bovine serum; Platelet lysate

Introduction

Since the work done by Zuk et al, when stem cells were isolated from human adipose tissue for the first time, the field of clinical research involving Adipose-Derived Mesenchymal Stromal Cells (ADSC) has grown and many studies have been conducted to explore the use of these cells in regenerative medicine and tissue engineering [1-7].

The methodological procedure for obtaining and isolating Mesenchymal Stromal/Stem Cells (MSC) is a complex process [3-6,8-11]. The methodology classically used for the isolation of ADSC is performed with the collagenase enzyme, but alternatives methods include the use of enzymes such as trypsin, dispase, and hyaluronidase or solutions containing combinations of more than one type of enzyme [12-14]. Even though the methodologies can vary regarding enzyme type, concentration, and incubation time, almost all are based on enzymatic digestion, which has some limitations. It has been shown that enzymes may contain substantial amounts of contaminants, xenoproteins, endotoxins, and other peptides, besides increasing the cost of the process [2,11,15,16].

The procedure for cell proliferation, in turn, requires the use of supplements to enrich the culture medium in order to sustain cell growth. The supplements of the culture media include growth factors, nutrients for proliferation and differentiation, as well as binding factors and compounds to inactivate toxic compounds [9,10,17,18]. To this end, 10 to 20% Fetal Bovine Serum (FBS) or calf serum is often used, even though these substances pose a risk to cells due to potential contamination by viruses, mycoplasmas, prions, or unidentified zoonotic agents that have a high content of xenogeneic proteins. These potential infectious agents may be associated with cultured cells and subsequently cause immunological reactions in the patient. Also, these cultured cells may contain unidentified substances that could cause interference, inhibiting growth or inducing cell differentiation [4,9-11,17,18].

Here we tested the mechanical dissociation, and cell cultivation with a lysate of platelets for the isolation and proliferation of hADSC, and compared these techniques with traditional methods using collagenase and fetal bovine serum, aiming to create a protocol for isolation and successful cell proliferation without xenobiotic components as collagenase and fetal bovine serum.

Objective

The aim of this study was to establish an efficient and reproducible protocol for the isolation and cultivation of human adipose-derived mesenchymal stromal cell that prescinds the use of xenobiotic products such as collagenase and fetal bovine serum while presenting similar cultivation results.

Materials and Methods

The adipose tissue used was obtained from two women in good general health, one 53 and the other 56 years old, who were submitted to abdominoplasty procedures. The material was donated by the Hospital Medical Care Institute (IAM) (Assis, São Paulo, Brazil) and the donors were informed about the research that would be done and signed the free and informed consent form. For the preparation of the platelet lysate, 4 bags of platelet concentrate were used.

The present experimental laboratory study was approved by the Research Ethics Committee of the São Paulo State University - UNESP, Campus Assis (SP, Brazil), under the Brazil Platform CAAE registration number 16624813.1.0000.5401.

Preparation of platelet lysate

The platelet lysate contained approximately 8.5 × 108 platelets/mL. Initially, the bags were subjected to four successive freezing processes at -80°C for 6 hours and thawing in a water bath at 37°C for 15 minutes to promote cell lysis.

Next, the lysate solutions were centrifuged 4 times at 3600 g for 30 minutes to remove the cell debris. Finally, the contents were filtered in 0.22 μm filters (Jet Biofil, São Paulo, Brazil) and frozen at -20°C until the time of use. At the time of preparation of the culture medium, 2 UI/mL of heparin was added.

Preparation of adipose tissue

After surgery, the obtained adipose tissue was placed in a sterile container containing phosphate buffer pH: 7.2 (PBS; LGC, São Paulo, Brazil) supplemented with 2% antimycotic-antibiotic (Penicillin, Streptomycin, and Fungizone; Gibco, New York, USA), and allowed to rest for 24 hours at 4°C for disinfection.

Next, the adipose tissue was divided into four groups as follows: cells treated with enzymatic digestion and cultured with FBS (ED+FBS); cells isolated by mechanical dissociation and cultured with FBS (MD+FBS); cells isolated by enzymatic digestion and cultured with a platelet lysate (ED+PL); and cells isolated by mechanical dissociation and cultured with a platelet lysate (MD+PL).

Adipose-derived mesenchymal stromal cells isolation

Enzymatic digestion: For the enzymatic digestion, the method proposed by Zuk et al. was used with minor modifications [1]. The adipose tissue was minced into small pieces and subjected to enzymatic digestion with collagenase type I (Gibco, New York, USA), at a concentration of 0.25%. Next, the enzyme was neutralized by the addition of Dulbecco’s Modified Eagle Medium F12 culture medium (DMEM-F12; Gibco, New York, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, New York, USA) or platelet lysate according to the study group. The resulting cell suspension was centrifuged for 10 minutes at 900 g. Finally, the cells were seeded in cell culture flasks (BD, NJ, USA) at a concentration of 1 × 105 cells/cm2 in DMEM-F12 culture medium (Gibco, New York, USA) supplemented with a 1% antibiotic-antimycotic solution (Penicillin, Streptomycin, and Fungizone; Gibco, New York, USA) and 10% FBS (Gibco, New York, USA) or 10% platelet lysate. Subsequently, the cells were incubated at 37°C and 5% CO2.

Mechanical dissociation: The adipose tissue was subjected to mechanical dissociation with the aid of two needles (BD, NJ, USA) folded into an L shape to separate the cells (FIG. 1). The medium containing the dissociated cells was placed in syringes that remained in an upright position for about 10 minutes. The collected material was centrifuged for 10 minutes at 900 g. The precipitate (pellet) obtained was re-suspended in 1 mL of culture medium and a 10 μL aliquot was removed for cell counting and determination of cellular viability. Finally, the cells were seeded in culture flasks (BD, NJ USA) at a concentration of 1 × 105 cells/cm2 in DMEM-F12 culture medium (Gibco, New York, USA) supplemented with a 1% antibiotic-antimycotic solution (Penicillin, Streptomycin, and Fungizone; Gibco, New York, USA) and 10% FBS (Gibco, New York, USA) or platelet lysate. Subsequently, the cells were incubated at 37°C and 5% CO2.

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Figure 1: Mechanical dissociation of stem cells derived from adipose tissue; A: Disinfecting tissue; B and C: Cleaning of the adipose tissue; D and E: Mechanical dissociation; F: Separation of cells and cellular debris by density difference; G: Filtering; H: Precipitate formation after centrifugation; I: Incubation in a CO2 incubator.

The growth of adipose-derived mesenchymal stromal cells (ADSC)

Cells cultured to the 5th passage were seeded in 96-well plates (BD, NJ, USA) in triplicate at a density of 1 × 103 cells/well in DMEM-F12 medium containing 10% FBS or 10% platelet lysate and 1% antibiotic-antimycotic solution (Gibco, New York, USA). Using the Vybrant MTT Cell Proliferation Assay Kit (Gibco, New York, USA) 2-(4.5-dimethyl-2-thiazole)-3.5-diphenyl bromide (MTT) tests were performed every 24 hours for 10 consecutive days, following the manufacturer’s instructions.

Based on the growth curve, the folding time of the population (PDT: Population Doubling Time) was calculated using the equation I, where t: time, N: final number of cells, and N0: initial number of cells as described by Arrigoni et al [19].

equation

Immunophenotyping

For immunophenotyping, the hADSC were analyzed by flow cytometry (5000 events) using the FACSCalibur (BD, New Jersey, USA). hADSC cultivated to the 5th passage were labeled with CD90, CD73, CD105, CD45, CD34, HLA-DR, CD19, and CD11b (BD, New Jersey, USA) antibodies and analyzed by the WinMDI 2.8 program.

Differentiation of stem cells derived from adipose tissue

To induce differentiation, hADSC cultivated to the 5th passage were maintained for 7, 14, and 21 days, respectively, in the presence of adipogenic, chondrogenic, and osteogenic StemPro differentiation kits (Gibco; New York, USA), according to the method proposed by the manufacturer. The adipogenic differentiation was confirmed by staining analysis with Oil Red O (Sigma-Aldrich, Missouri, USA). The chondrogenic differentiation was confirmed by staining analysis with Alcian Blue (Sigma-Aldrich, Missouri, USA) and chondrogenic differentiation was confirmed by staining with Alizarin Red S analysis (Sigma-Aldrich; Missouri, USA).

Results

In order to establish a protocol for hADSC isolation and cultivation that does not use xenobiotic components such as collagenase and Fetal Bovine Serum (FBS), cells were isolated by mechanic dissociation and then cultivated in the presence of human platelet lysate (PL). As a control for comparison, enzymatic digestion (using collagenase) was also performed in another set of cells, as well as the cultivation in the presence of FBS.

Cells isolated by either technique adhered to the surface of culture flasks after two days of incubation. Between the third and fifth day, cells began developing a fibroblastoid aspect and forming the first colonies (FIG. 2A).

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Figure 2: Human adipose-derived mesenchymal stem cells culture characteristics; A: Fibroblastoid morphology of hADSC in culture; B: Culture with high confluence (>80%) of cells and a homogeneous monolayer aspect.

In the primary culture, red blood cells and mature adipocytes were observed in both cell cultures, with the highest incidence in cultures isolated by mechanical dissociation. These cells were eliminated during the exchange of media and dissociation with Tryple (Gibco, New York, USA), so that in the third passage, all cultures showed an aspect of homogeneous monolayer of cells, similar in morphology to the fibroblasts that were maintained until the end of the culture when we acquired enough cells (between 107 and 108 cells per Kg) (FIG. 2B).

The confluence of 70% to 80% that allows cells to be seeded into new culture flasks was observed on day 8 for the cultures isolated by enzymatic digestion and on the 10th day for the cultures isolated by mechanical dissociation. The other passages took between 4 and 5 days of culture for cells grown with either technique. All cultures were maintained until the 5th passage.

To investigate the potential of adhesion and proliferation of hADSC isolated by Mechanical Dissociation (MD) and Enzymatic Digestion (ED), and then cultured in medium supplemented with FBS or PL, the MTT readings were performed, with growth curves lasting 10 days. The mean growth curves are illustrated in FIG. 3.

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Figure 3: Growth kinetics of hADSC isolated by enzymatic digestion (ED) or mechanical dissociation (MD) and cultured with fetal bovine serum (FBS) or platelet lysate (PL); A: ED+FBS=Cells isolated by enzymatic digestion (ED) and cultivated with fetal bovine serum (FBS); MD+FBS=Cells isolated by Mechanical Dissociation (MD) and cultured with FBS; B: ED+PL=Cells isolated by enzymatic digestion and cultured with a platelet lysate (PL); MD+PL=Cells isolated by mechanical dissociation and cultured with a platelet lysate.

Initially, all groups showed a growth phase (lag phase) that represents the metabolic adaptation period of the cells after plating, when there is adhesion, elongation, and production of the components necessary for cell expansion. In the group isolated by mechanical dissociation and cultivated with platelet lysate (MD+PL) this phase was observed only during the first 24 hours of cultivation, while in the group isolated by mechanical dissociation and cultivated with SBF (MD+FBS) and in groups isolated by enzymatic digestion (ED+PL and ED+FBS), the lag phase extended to the first 48 hours after plating (FIG. 3).

Subsequent to this initial growth period, the cells exhibited an exponential growth phase (log phase). During this phase, the groups showed two different curve patterns. As can be observed in FIG. 3, cells grown in culture medium supplemented with platelet lysate (ED+PL and MD+PL) presented a higher initial growth compared to the groups supplemented with FBS (MD+FBS and ED+FBS). However, the log phase of ED+PL and MD+PL groups was maintained for only 6 and 5 days, respectively, while the MD+FBS and ED+FBS groups, though with a slower growth, maintained the cell proliferation for up to 8 and 9 days, respectively. No statistical differences in the log phase of the ED+PL and MD+PL groups were observed; in the groups cultivated with FBS, a higher growth was observed MD+FBS group in comparison with the ED+FBS group (statistical difference at days 2, 5, 6, 7, 8 and 9). In regard to the groups isolated enzymatic digestion (ED+PL and ED+FBS), the FBS group presented a lower growth curve, and a growth peak at day 8, while the PL group peaked at day 3 (statistical difference at days 3 to 7). In addition, the ED+FBS group presented the lowest growth curve amongst all groups. The groups isolated by mechanical dissociation (MD+PL and MD+FBS) presented similar growth curves, with different growth kinetics and peaks, at days 5 and 8, respectively. Due to this divergence, they differed at days 2 to 9. The complete statistical analysis can be found in TABLE 1. After the log phase, none of the groups showed a stationary phase, and all entered the decline phase.

Day One-Way ANOVA Post HOC (LSD)
ED+FBS × DM+FBS ED+FBS × ED+PL ED+FBS × DM+PL DM+FBS × ED+PL DM+FBS × DM+PL ED+PL × DM+PL
1 F(3,8)=3.606. p=0.065 - - - - - -
2 F(3,8)=68.934. p ≤ 0.001 p=0.003 - p ≤ 0.001 p=0.033 p ≤ 0.001 p ≤ 0.001
3 F(3,8)=17.598. p=0.001 - p ≤ 0.001 p=0.001 p=0.001 p=0.018 -
4 F(3,8)=13.929. p=0.002 - p=0.001 p=0.001 p=0.008 p=0.004 -
5 F(3,8)=153.744
p ≤ 0.001
p=0.003 p ≤ 0.001 p ≤ 0.001 p ≤ 0.001 p ≤ 0.001 -
6 F(3,8)=84.338. p ≤ 0.001 p=0.001 p ≤ 0.001 p ≤ 0.001 p ≤ 0.001 p=0.002 p=0.001
7 F(3,8)=13.284. p=0.002 p=0.001 p=0.001 - - p=0.009 p=0.008
8 F(3,8)=8.893. p=0.006 p=0.003 - - p=0.010 p=0.002 -
9 F(3,8)=7.153. p=0.012 p=0.015 - - p=0.009 p=0.002 -
10 F(3,8)=1.659. p=0.252 - - - - - -

Table 1: Complete statistical analyses of growth kinetics of hADSC isolated by enzymatic digestion (ED) or mechanical dissociation (MD) and cultured with fetal bovine serum (FBS) or platelet lysate (PL); ED+FBS=Cells isolated by enzymatic digestion (ED) and cultivated with fetal bovine serum (FBS); MD+FBS=Cells isolated by mechanical dissociation (MD) and cultured with FBS; ED+PL=Cells isolated by enzymatic digestion and cultured with a platelet lysate (PL); MD+PL=Cells isolated by mechanical dissociation and cultured with a platelet lysate.

With respect to the population doubling time (PDT), which represents an important marker of proliferative capacity of cells, it was observed that the average PDT (TABLE 2) reflected a similar behavior to that observed in the growth curves (FIG. 2). The most remarkable aspect in regard to the PDT refers to the comparison between groups cultivated with FBS and PL, where FBS groups presented a slower growth pattern, shown by a higher PDT in relation to the PL groups (TABLE 1).

Group PDT (hours)
ED+FBS 59.59
MD+FBS 49.75
ED+PL 39.96
MD+PL 20.69

Table 2: Population Doubling Time (PDT) of the adipose-derived stem cell isolated and cultured following different protocols. ED+FBS=Single cells treated with enzymatic digestion (ED) and cultivated with fetal bovine serum (FBS); MD+FBS=Cells isolated by mechanical dissociation (MD) and cultured with FBS; ED+PL=Cells isolated by enzymatic digestion and cultured with a platelet lysate (PL); MD+PL=Cells isolated by mechanical dissociation and cultured with a platelet lysate.

After isolation and purification of hADSC, surface markers were analyzed to evaluate the efficiency of separation (TABLE 3). Cells cultured with platelet lysate show, for different markers, values close to those recommended by the International Society for Cellular Therapy (ISCT), as reported by Dominici et al [20]. These results indicate a low rate of contamination by other cell types. However, samples grown with fetal bovine serum showed values lower than 95% for CD73/CD105 and CD90/CD105 (TABLE 3) and, therefore, the percentage values of the markers obtained in the experimental groups grown with FBS were lower than those proposed by the ISCT [18].

Markers ED+FBS MD+FBS ED+PL MD+PL
CD73/CD90 98.39% 88.07% 99.62% 93.12%
CD73/CD105 64.43% 79.82% 97.11% 92.50%
CD90/CD105 60.46% 77.98% 98.93% 98.12%
CD34 0% 0% 0% 0%
CD11b 0.10% 1.17% 0.06% 1.05%
HLA-DR 0.80% 1.79% 0.30% 0%
CD19 0% 0.39% 0.10% 0%
Total ADSC 60.46% 77.98% 97.11% 92.50%

Table 3: Characterization of adipose-derived stem cells by flow cytometry; ED+FBS=Single cells treated with enzymatic digestion and cultured with fetal bovine serum (FBS); MD+FBS=Cells isolated by mechanical dissociation and cultured with FBS; ED+PL=Cells isolated by enzymatic digestion and cultured with a platelet lysate; MD+PL=Cells isolated by mechanical dissociation and cultured with a platelet lysate.

The differentiation potential of hADSC into adipocytes, chondrocytes, and osteocytes was investigated to determine whether the cells isolated by different methods meet the basic requirements for being defined and characterized as mesenchymal stem cells. According to the results shown in FIG. 4, all groups demonstrated the differentiation potential of the three lineages. The adipogenic differentiation was confirmed by the presence of lipid vacuoles indicated by staining with Oil Red O. The osteogenesis was verified by staining the bone matrix with Alizarin Red S. Finally, the chondrogenic differentiation was confirmed with Alcian Blue dye. Blue indicates the synthesis of proteoglycans by chondrocytes.

Discussion

The ADSC transplantation represents a promising and emerging therapeutic alternative with great potential for the repair and regeneration of organs and tissues in acute or chronic injuries [6,21,22].

The present study aimed to compare the efficacy of cell isolation and cultivation in the absence of xenobiotic compounds with the traditionally used methods, which rely on collagenase for enzymatic dissociation and SBF for cell cultivation. The results presented herein allow the proposition that the mechanical dissociation allows to carry out hADSC cultures with the same efficiency in the proliferation and cell viability obtained from the enzymatic digestion method, without the possible risks inherent in the use of xenobiotic components. Other authors, such as Bianchi et al and Shah et al, also proposed alternative techniques for isolating ADSC that prescind the use of enzymes and reported results similar to those obtained in the present study [22,23]. In a more recent research, Tozetti and colleagues reported efficient cell proliferation using different xeno-free protocols.

In the work of Shah et al, the proposed method is based on lipoaspirate washing with a phosphate buffer and subsequent centrifugation of the supernatant [23]. As a similar method used by Bianchi et al, the authors proposed an enclosed apparatus for immersion of the lipoaspirate, containing small stainless steel balls which, under stirring, gradually break up the tissue, thereby releasing the ADSC [22]. Although these are different techniques than the ones presented in this study, they all indicate that the isolation of ADSC can be accomplished without the need of enzymes, and that there are viable and feasible alternatives that produces reproducible results and matches the recommendations issued by the National Health Surveillance Agency in Brazil (ANVISA) (Directory Board Resolution-RCD No. 9 of March 14, 2011, ANVISA),which endorse that the use of xenobiotic products as collagenase and FBS in cell culture intended for use in cell therapy procedures in human patients should be avoided or minimized [24,25].

The growth kinetics (FIG. 3) and PDT (TABLE 2) regarding the non-enzymatic methods are discordant with those described by Shah et al, as these authors observed that cells isolated by the lipoaspirate washing method presented a proliferation capacity 19 times lower than the rate found in cells isolated by collagenase [23]. The same does not occur with the mechanical dissociation method proposed in the present study, which exhibits similar growth kinetics to that of enzymatic digestion.

The use of platelet lysate as a supplement to the culture medium also altered cell growth, resulting in a faster proliferative rate than that found in the groups supplemented with FBS (FIG. 3). These observations are consistent with those found by several other authors who also described very similar results when comparing ADSC growth cultured in medium supplemented with 10% FBS or PL [8,25-27].

After analyzing the proliferative capacity of cells isolated and cultured under the different conditions proposed, cell characterization was performed (immunophenotype) by flow cytometry. The results (TABLE 3) showed that cells cultured with platelet lysate demonstrate low levels of contamination by other cell types, taking into account the proposals of the International Society for Cellular Therapy (ISCT) for characterizing mesenchymal stem cells where CD105, CD73, and CD90 markers are present in percentages above 95%, while the cells for CD34, CD11b, CD19, and HLA-DR surface antigens should present in percentage values below 2% [20]. More recently, the ISCT established more specific guidelines for the characterization of mesenchymal stem cells isolated from adipose tissue, determining that the CD105, CD90, and CD73 markers should be present in over 70% of the cells12; this means that the MD+FBS group is also among the groups that meet the ISCT criteria [12]. Thus, the only group that did not reach the percentage proposed by the ISCT was the ED+FBS group, which showed positive staining of 64.43% for CD73/CD105 and 60.46% for CD90/CD105. Similar results were also reported by other research groups [28-30], which described the reduction of CD73 and CD105 markers when cells isolated by collagenase and cultured with FBS were analyzed after the 5th passage. The same may have occurred in the present study since the cells were only examined on the 6th passage. It is also important to mention that, among the 4 groups evaluated in the present study, the growth curve observed for the ED+FBS was the lowest, while the other 3 groups, although presenting different kinetics, showed the comparable total number of cells at their peak.

Finally, to complete the cell characterization, cells were induced to differentiate into adipogenic, osteogenic, and chondrogenic lineages according to the criteria recommended by the ISCT and adopted by different authors [20,25,27,28,30,31]. Groups grown either with FBS or platelet lysate presented the differentiation capacity of the chondrogenic, osteogenic, and adipogenic lineages (FIG. 4). This result allows the affirmation that the cells used for the experiments were indeed of the mesenchymal type.

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Figure 4: Differentiation of adipose-derived stem cells into adipocytes, chondrocytes, and osteocytes.

Conclusion

The results obtained in the present study allow the conclusion that the isolation of hADSC by mechanical dissociation and the use of platelet lysate represent a viable, reproducible and low-cost methodological alternative. In addition, the isolation of hADSC in xeno-free conditions showed equivalent results in relation to the classical methodology with the use of collagenase and FBS. Further, the methodology proposed in this study may represent a potentially promising alternative suitable in cell-based therapies in different pathological conditions of human patients.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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