Cell and Tissue Banking

, Volume 14, Issue 1, pp 65–76

Comparison of vitrification and slow cooling for umbilical tissues

Authors

  • Lilian Da-Croce
    • Laboratory of Animal and Human Genetics, Department of General BiologyUniversidade Federal de Minas Gerais
    • Sector of Cytogenetics, Department of GeneticsHermes Pardini Institute
    • Sector of Cytogenetics, Department of GeneticsHermes Pardini Institute
  • Patrícia Caroline Angelo
    • Sector of Cytogenetics, Department of GeneticsHermes Pardini Institute
  • Eduardo Alves Bambirra
    • Department of Pathological Anatomy and Legal Medicine, Faculty of MedicineUniversidade Federal de Minas Gerais
    • Department of Pathological AnatomyHermes Pardini Institute
  • Antônio Carlos Vieira Cabral
    • Department of Ginecology and Obstetrics, Faculty of MedicineUniversidade Federal de Minas Gerais
  • Ana Lúcia Brunialti Godard
    • Laboratory of Animal and Human Genetics, Department of General BiologyUniversidade Federal de Minas Gerais
Original Paper

DOI: 10.1007/s10561-012-9301-9

Cite this article as:
Da-Croce, L., Gambarini-Paiva, G.H.R., Angelo, P.C. et al. Cell Tissue Bank (2013) 14: 65. doi:10.1007/s10561-012-9301-9

Abstract

The tissue cryopreservation maintains the cellular metabolism in a quiescence state and makes the conservation possible for an indefinite period of time. The choice of an appropriate cryopreservation protocol is essential for maintenance of cryopreserved tissue banks. This study evaluated 10 samples of umbilical cord, from which small fragments of tissue (Wharton’s jelly and cord lining membrane) were subjected to two protocols of cryopreservation: slow cooling and vitrification. The samples were frozen for a period of time ranging from 5 to 78 days. The efficiency of cryopreservation was evaluated by testing cell viability, histological analysis, cell culture, cytogenetic analysis and comparison with the results of the fresh samples. The results showed that the slow cooling protocol was more efficient than the vitrification for cryopreservation of umbilical cord tissue, because it has caused fewer changes in the structure of tissue (edema and degeneration of the epithelium) and, despite the significant decrease cell viability compared to fresh samples, the ability of cell proliferation in vitro was preserved in most samples. In conclusion, this study showed that it is possible to cryopreserve small fragments of tissue from the umbilical cord and, to obtain viable cells capable of proliferation in vitro after thawing, contributing to the creation of a frozen tissue bank.

Keywords

CryopreservationVitrificationSlow coolingTissueHuman umbilical cord

Introduction

The human umbilical cord is a helical body that makes the connection between the mother (via the placenta) and the baby, being the most important part of the fetal-placental, playing a role in gas exchange and baby’s nutrition (Naro et al. 2001). It consists of two arteries and one vein surrounded by Wharton’s jelly and covered by an outer layer of amniotic membrane, also called cord lining membrane (subamniotic region) of the umbilical cord (Kita et al. 2010). Mesenchymal stem cells were isolated from different compartments of the umbilical cord, like from the epithelium and subendothelium of the umbilical vein, arteries and perivascular area of the umbilical vein, as well as Wharton’s jelly and cord lining membrane (Covas et al. 2003; Sarugaser et al. 2005; Weiss and Troyer 2006; Secco et al. 2008; Ishige et al. 2009; Kita et al. 2010). For the therapeutic usages of these stem cells to become possible, it is necessary to have an efficient method of cryopreservation, which keeps preserved the viability and potential of these cells.

The two basic approaches to cryopreservation is vitrification and slow cooling (Karlsson and Toner 1996). Vitrification is a rapid freezing process, based on fast cooling rates under high concentrations of cryoprotectants. In literature, there are two main strategies for vitrification. In the first method, samples are placed into the straws for vitrification, which allows the achievement of high rates of cooling and heating (Huang et al. 2005; Keros et al. 2009; Curaba et al. 2011). The second strategy is used for large sample size (when they do not fit into the straws along with the cryoprotectant solutions), and consists in the use of very high concentrations of cryoprotectants, which results in slower rates of cooling and heating (Fahy et al. 2004; Song et al. 2004). The straws or tubes with the samples and cryoprotectant solutions are then subjected to cryogenic temperatures, usually immersed directly into liquid nitrogen (Kuleshova et al. 2007). The cryopreservation solutions and the samples are often kept around 4 °C for the vitrification procedures, which decreases the cellular cytotoxicity (Kasai et al. 1990). The advantage is that vitrification induces a glasslike solidification of living cells that completely avoids the formation of ice crystals, which are able to damage the cells during freezing and thawing (Kuleshova and Lopata 2002). However, high concentrations of cryoprotectants, which are associated with chemical toxicity and osmotic shock, can be harmful to the cells, leading to a low-efficiency process of maintaining cell viability after thawing (Kuleshova and Lopata 2002; Luciano et al. 2009; Kim et al. 2011). On the other hand the slow freezing requires low concentrations of cryoprotectants and programmed freezing of the samples by using controlled freezing chambers and cooling rates that decrease gradually (Kuleshova and Lopata 2002). This low concentration of cryoprotectant used in slow cooling is an advantage, however, the fact that the freezing process occurs gradually does not efficiently avoid the formation of ice crystals, which is considered a disadvantage (Luciano et al. 2009).

Even though several strategies for cryopreservation of cells and tissue have already been described (Meryman and Kafig 1955; Rowe 1996; Takahashi et al. 1988; Valeri et al. 2000; Fahy et al. 2004; Agudelo and Iwata 2008), the available protocols for cryopreservation may not be fully adequate for the target tissue, often resulting in low recovery rates of viable tissue (Kuleshova and Lopata 2002; Luciano et al. 2009; Kim et al. 2011; Oskam et al. 2011). Taking into consideration that different factors can influence the success of cryopreservation, including the components of the cryopreservation media, the nature of cryoprotectants, the freezing process, the rate of cooling, the thawing process and the intrinsic susceptibility of cells to be damaged during the freezing process (Mazur 1984; Hengstler et al. 2000; Ieropoli et al. 2004; Son et al. 2004; Miyamoto et al. 2006; Meryman 2007; Murua et al. 2009), and still do not have a well-established protocol for the cryopreservation of umbilical cord tissue, this study tested two protocols of cryopreservation, slow cooling and vitrification for the cryopreservation of this type of tissue. In addition, the effectiveness of these methods we evaluated for maintaining the integrity, tissue morphology and structure, cell viability, the ability of cell to proliferate in vitro, as well as the ability to induce chromosomal alterations, in order to establish a cryopreservation method suitable for future implementation of banks of cryopreserved umbilical cord tissue.

Materials and methods

Sample collection and experimental design

Ten samples of umbilical cord were collected from patients who had cesarean or natural delivery, term delivery without pregnancy complicating factors, from the Hospital das Clinicas, UFMG, Belo Horizonte—MG. The collections were also performed in the operating room soon after birth, without offering risks or discomfort for the mother and/or the baby. All participants signed an informed consent. This study was approved by the Ethics Committee of Universidade Federal de Minas Gerais (COEP), No 0326.0.203.000-10.

From each umbilical cord, four fragments of approximately 10 cm were collected. The samples were placed in sterile containers, properly identified, containing saline and transported at 4 °C to the laboratory. The samples were dissected macroscopically, also sterile, to obtain small fragments of approximately 5 × 2 × 2 mm. In total 138 fragments were obtained per sample, with 69 fragments dissected from Wharton’s jelly and 69 from the cord lining membrane of the umbilical cord. The dissected fragments of each tissue were arranged in three groups, each containing 23 fragments. For each sample, a group of Wharton’s jelly and a group of cord lining membrane were subjected to slow cooling, another group of each tissue was subjected to the vitrification process and the third group of each tissue served as an experimental control (fresh). The fresh samples and those submitted to slow cooling and vitrification, after being thawed, were submitted to histological study, evaluation of cell viability, cell culture and cytogenetic analysis (Fig. 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs10561-012-9301-9/MediaObjects/10561_2012_9301_Fig1_HTML.gif
Fig. 1

Experiment outline. From each umbilical cord 69 Wharton’s jelly and 69 cord lining membrane fragments have been dissected. The fragments of each tissue were arranged in three groups, each containing 23 fragments. One group as control (fresh tissue), another group was subjected to slow cooling and the other to the vitrification process. After thawing the samples, three fragments from each group (including the control group) were sent for histological analysis. The remaining 20 fragments were used to assess cell viability, cell culture and cytogenetic analysis

Slow cooling

The slow cooling process was carried out as described by Yu-bin et al. (2007), with modifications. The fragments (Wharton’s jelly and cord lining membrane) were initially incubated for 15 min in base medium: RPMI 1640 (Invitrogen, Grand Island, NY, USA) supplemented with 20 % fetal bovine serum (Invitrogen, Grand Island, NY, USA). After that time, they were immersed for 5 min in cryo-solution 3 (1.5 mol/L of DMSO diluted in basic medium) and cooled down to 4 °C. Thereafter, each fragment was transferred to a cryotube containing 1 mL of cryo-solution 4 (1.5 mol/L DMSO and 0.1 mol/L sucrose diluted in basic medium) and cryovials were taken to the Cryomed Controlled Rate Freezer (Thermo Scientific, Waltham, MA, USA). The program consisted of keeping the samples at 4 °C for 30 min, reducing the temperature from 4 to −8 °C at −2 °C/min, maintaining at −8 °C for 10 min, then cooling down again from temperature of −8 to −40 °C at −0.3 °C/min and then down to −150 °C at −30 °C/min. After the program was finished, the cryovials were stored in the tank of liquid nitrogen for periods ranging from 5 to 78 days.

Vitrification

The vitrification process was also performed according to the method described by Yu-bin (2007), with modifications. All cryoprotectant solutions were cooled down to a temperature of 4 °C before being added to the tissue. The fragments (Wharton’s jelly and cord lining membrane) were placed for 15 min in the base medium at 4 °C. After that, they were incubated for 5 min in a cryo-solution 1 cooled down to 4 °C: 2.0 mol/L DMSO (Baxter, Bioniche Teoranta, Galway, Ireland) and 0.1 mol/L sucrose (Merck, Darmstadt, Germany) diluted in base medium. They were then transferred to cryo-solution 2: 2.0 mol/L DMSO, 2.0 mol/L propanediol (Merck, Darmstadt, Germany) and 0.2 mol/L sucrose diluted in base medium, cooled down to 4 °C maintained for 5 min. Each fragment was then removed from the solution and immediately plunged into liquid nitrogen, being then removed with cooled forceps, placed in cryovials (Corning Incorporation, NY, USA) and stored in liquid nitrogen tank for periods of time ranging from 5 to 78 days.

Thawing of the samples

The thawing process was the same for the samples submitted to slow cooling and vitrification. The cryovials were removed from liquid nitrogen and immediately placed in a water bath at 37 °C for about 7 min. Soon after the samples were washed three times with base medium.

Histological analysis

A cross section of approximately 5 cm was obtained from each umbilical cord and fixed in neutral formalin. Besides the cross-section, three fragments from Wharton’s jelly and three from cord lining membrane of each fresh umbilical cord, post-vitrification and post-slow cooling were also fixed in neutral formalin. The fixed samples were embedded in paraffin, and from 5 μm cuts slides were prepared slides and stained with hematoxylin-eosin for histological analysis performed by experienced pathologist. A cross section of each cord was first evaluated and taken as reference for comparison with the fragments of Wharton’s jelly and cord lining membrane after cryopreservation processes.

Assessment of cell viability and cell culture

The assessment of cell viability by Trypan Blue method was performed immediately before the entry into cell culture. The time between sample collection and processing analysis of cell viability and cell culture ranged from 2 to 18 h.

For each umbilical cord, both Wharton’s jelly and the cord lining membrane, the 20 fragments from each of the three groups (fresh, post-vitrification and post-slow cooling) were digested with collagenase type II (3 mg/mL, Invitrogen, Grand Island, NY, USA) for about 3 h. A counting of viable cells were performed using Trypan blue in a Neubauer chamber, from an aliquot of 10 mL of the packed cell obtained from each sample. The rest of the digested material was subjected to cell culture. The cell cultures were performed in T25 flasks (Greiner Bio-One, Brazil) using 5 mL of culture medium Amniomax (Invitrogen, Grand Island, NY, USA), incubated at 37 °C in a CO2 incubator (Thermo model 3111), supplemented as recommended by the manufacturer, being the culture medium changed every 4 days. When necessary, treatment with 0.25 % Trypsin EDTA (Invitrogen, Grand Island, NY, USA) was performed to promote cell expansion in the bottle, but no cell culture was under cultivated. The cell growth was monitored daily under inverted light microscopy and, after reaching 80 % confluence (80 % of the cultivable area of the bottle covered by cells), were sent to a karyotype preparation.

Cytogenetic analysis

To obtain chromosomes hypotonization was performed with 0.075 M KCl (Merck, Darmstadt, Germany) and fixation was made with methanol/acetic acid (Merck, Darmstadt, Germany). An average of five slides per case were prepared, incubated overnight at 55 °C and subsequently held a GTG banding with trypsin solution diluted in Dulbecco’s buffer (Invitrogen, Grand Island, NY, USA) at a ratio of 1:250 and Giemsa staining (Gustashaw 1997). Chromosomal analysis was performed using an optical microscope (Nikon E400) coupled to a digital system and a karyotyping analysis software (Applied Spectral Imaging, version 6.0). The results of the karyotype followed the ISCN 2009 recommendations.

Statistical analysis

The results were analyzed using Prism software (version 4.0, GraphPad Software Inc., CA, USA). Paired Student’s t tests and correlation (r Pearson) were performed in the statistical analysis results of cell viability and culture period. Mean values and standard deviations were presented as mean ± standard deviation. P < 0.05 was considered statistically significant.

Results

The samples were frozen for a period of time ranging between 5 and 78 days, averaging 19.60 ± 21.96 days. The efficiency of cryopreservation’s was evaluated by testing cell viability, histological analysis, cell culture, cytogenetic analysis and comparison with the results from the fresh samples.

Histological analysis

Histological analysis of the umbilical cord, after the cryopreservation process, showed interstitial edema both in the Wharton’s jelly and in the cord lining membrane. The latter also showed epithelial degeneration as a result of intracellular edema in the epithelial cells (vacuoles). The edema was classified as mild (1+), moderate (2+), intense (3+) or very intense (4+). For the Wharton’s jelly, the 10 fresh samples showed normal morphology. After slow cooling, only 2/10 samples showed very intense interstitial edema (4+) and after vitrification, the majority (9/10) showed interstitial edema (3+) or very intense (4+) (Table 1, Fig. 2). For the cord lining membrane, 2/10 fresh samples showed very intense interstitial edema (4+) and vacuole in epithelial cells, culminating in the total degeneration of the epithelium, while the other samples maintained their normal morphology. After both slow cooling and vitrification cryopreservation process all the samples from the cord lining membrane showed interstitial edema, ranging from moderate (2+) to very intense (4+), and vacuole in epithelial cells, leading to degeneration of the epithelium (Table 1, Fig. 2).
Table 1

Results of cell viability, cell culture period, histological and cytogenetic analysis for the tissue of the umbilical cord, Wharton’s jelly and cord lining membrane, divided into three groups: fresh (control), post-slow cooling and post-vitrification

Samples

Fresh

Post-slow cooling

Post-vitrification

Stay at −196 °C (day)

Histology

Viability (%)

Culture (days)

Karyotype

Histology

Viability (%)

Culture (days)

Karyotype

Histology

Viability (%)

Culture (days)

Karyotype

Wharton’s jelly

1

N

50

20

46,XY[30]

N

33

26

46,XY[30]

4+

0

78

2

N

67

N

50

17

46,XY[30]

4+

0

30

3

N

57

14

46,XX[30]

N

33

31

46,XX[5]

3+

0

17

4

N

75

21

46,XX[30]

4+

50

4+

0

12

5

N

83

14

46,XY[26]

4+

33

4+

0

12

6

N

60

12

46,XY[30]

N

33

25

46,XY[30]

4+

0

5

7

N

50

13

46,XY[30]

N

33

15

46,XY[30]

3+

0

5

8

N

67

11

46,XX[30]

N

75

20

46,XX[30]

N

0

21

9

N

50

13

46,XY[30]

N

66

20

92,XXYY[30]

4+

0

9

10

N

67

21

46,XX[30]

N

60

19

46,XX[30]

4+

0

7

Mean

 

63,55

15,44

  

46,73

21,63

     

19,60

SD

 

10,46

4,03

  

16,05

5,29

     

21,96

Cord lining membrane

1

N

69

9

46,XY[30]

*4+

33

30

46,XY[2]

*4+

0

78

2

N

63

26

46,XY[30]

*4+

33

14

46,XY[30]

*4+

0

30

3

N

88

11

46,XX[30]

*4+

40

14

46,XX[30]

*4+

0

17

4

*4+

50

19

46,XX[30]

*4+

0

*4+

0

12

5

*4+

100

15

46,XY[30]

*4+

50

24

46,XY[22]

*4+

0

12

6

N

63

14

46,XY[30]

*3+

50

15

46,XY[30]

*3+

0

18

46,XY[14]

5

7

N

57

11

46,XY[30]

*2+

50

14

46,XY[30]

*3+

0

28

92,XXYY[14]

5

8

N

63

11

46,XX[30]

*3+

57

25

46,XX[30]

*3+

33

28

46,XX[30]

21

9

N

88

9

46,XY[30]

* 4+

67

17

46,XY[20]

*4+

0

9

10

N

67

11

46,XX[30]

*4+

63

15

46,XX[14]

*4+

0

7

Média

 

70,51

13,60

  

44,29

18,66

  

3,33

24,66

 

19,60

SD

 

15,83

5,32

  

19,19

6,04

  

10,43

5,77

 

21,96

SD standard deviation, – Absence of cell growth, N normal morphology, and 2+: moderate interstitial edema, and 3+: intense interstitial edema, and 4+: very intense interstitial edema * presence of vacuoles epithelial cells, culminating degeneration of the epithelium

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Fig. 2

Histology of the umbilical cord tissue. a fresh Wharton’ Jelly presenting normal morphology, b Wharton’s Jelly after vitrification showing intense degree of interstitial edema (3+) c Wharton’s Jelly after slow cooling showing very intense interstitial edema (4+), d cord lining membrane featuring fresh normal morphology and preservation of the epithelium, e cord lining membrane after slow cooling showing moderate interstitial edema (2+) and degeneration of the epithelium showing detachment of the same subamniotic region; f cord lining membrane after slow cooling showing very intense interstitial edema (4+) and epithelial degeneration and disintegration. a, b and c: increase of ×200, d, e and f: an increase of ×100

Cell viability

The average of cell viability was 63.55 % ± 10.46 for fresh samples of Wharton’s jelly and 70.51 % ± 15.83 for fresh samples of the cord lining membrane. A significant decrease in the percentage of viable cells after slow cooling process for both Wharton’s jelly (46.73 % ± 16.05, P = 0.0119) and cord lining membrane (44.29 % ± 19.19, P = 0.0016) samples was observed (Fig. 3). After the vitrification process, viable cells were not detected in Wharton’s jelly samples. Only in a sample of the cord lining membrane viable cells were detected after vitrification, showing 33 % viability (Table 1).
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Fig. 3

Slow cooling effect on cell viability of the tissue from the umbilical cord. Significant P value (*) was obtained by paired Student’s t test, showing significant decrease in cell viability of Wharton’s jelly after slow cooling (B) compared to fresh Wharton’s jelly (A) and cord lining membrane post-slow cooling (D) in relation to the fresh cord lining membrane (C)

Cell culture

Although there was significant decrease in cell viability after slow cooling, the ability of proliferating in cell culture has been preserved in most samples. For the Wharton’s jelly, eight post-slow cooling samples and nine fresh samples showed growth in culture and taking from 15 to 31 days (21.63 ± 5.29) and from 11 to 21 days (15.44 ± 4.03), respectively, to reach 80 % confluence (Table 1). The comparison of culture period, reaching 80 % confluence, showed significant difference (P = 0.0219) before (fresh) and post-slow cooling of Wharton’s jelly for seven paired samples (Fig. 4). For the cord lining membrane, nine post-slow cooling samples and 10 fresh samples showed cell growth, taking from 14 to 30 days (18.66 ± 6.04) and from 9 to 26 days (13.60 ± 5.32), respectively, to reach 80 % confluence (Table 1). The comparison of culture period showed no significant difference before (fresh) and after slow cooling of the cord lining membrane, for the nine paired samples (Fig. 4). After vitrification, no Wharton’s jelly samples grew in culture and three samples of the cord lining membrane showed cell growth, taking from 18 to 28 days (24.66 ± 4.44) to obtain 80 % confluence.
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Fig. 4

Distribution of culture period to obtain 80 % confluence. A) Fresh Wharton jelly (n = 9), B) Wharton’s Jelly post-slow cooling (n = 8), C) Fresh cord lining membrane (n = 10), D) cord lining membrane post-slow cooling (n = 9), E) cord lining membrane post-vitrification (n = 3). P value obtained by paired Student’s t test, showed significant increase (*) of culture period of Wharton’s jelly after slow cooling compared to fresh

Cytogenetic analysis

For each cell culture medium were analyzed about 25 metaphases in resolution of 250–400 bands. Cytogenetic analysis of Wharton’s jelly might be held to nine fresh samples and eight post-slow cooling. All samples presented a normal karyotype result, except for one post-slow cooling sample (sample 9), which showed a tetraploid karyotype (92,XXYY) (Table 1). After vitrification, there was no culture growth in any of Wharton’s jelly sample to perform karyotyping.

For the cord lining membrane, it was possible to analyze 10 fresh samples and nine post-slow cooling ones. All samples presented a normal karyotype result (Table 1). After vitrification, only three samples showed cell growth and could be analyzed. One of these samples (sample 7) showed a tetraploid karyotype (92,XXYY), the others had normal karyotypes.

Discussion

This study tested the slow cooling and the vitrification process for cryopreservation of the following tissue of the umbilical cord: Wharton’s jelly and cord lining membrane. Considerable differences in the results were observed. The vitrification has led to morphological changes in the tissue, absence of viable cells in culture and growth in most of the samples. The slow cooling process proved to be the most effective method causing fewer changes in the tissue structure, higher rates of cell viability and better results in cell culture.

Histological analysis revealed the presence of moderate (2+), intense (3+) or very intense (4+) interstitial edema (abnormal accumulation of fluid within or between cells) in the umbilical cord tissue after the process of cryopreservation and vacuole (or intracellular edema) in the epithelial cells, leading to epithelial degeneration of the lining membrane samples after cryopreservation process. In vivo, the edema can be caused by an increase in intravascular hydrostatic pressure, changes in oncotic pressure (pressure exerted by plasma proteins), obstruction of lymphatic drainage or an increase in capillary permeability. The latter can be caused, among others, by toxic substances to cells (Metze 2004), such as cryoprotectants (sucrose, DMSO and propanediol) used in this study.

Thus, it is possible that cryoprotectants have contributed to the increased capillary permeability in the tissue, leading to the formation of edema. Only the epithelial cells present in the cord lining membrane showed vacuoles after the process of cryopreservation. Probably the structure of the umbilical cord with the presence of collagen and elastin (Kuhnel 2005) can protect Wharton’s jelly and subamniotic region fibroblastic cells from the formation of intracellular edema, while the cells of the thin cord lining membrane epithelium (Kuhnel 2005), do not have such protection, may be more prone to vacuoles formation of and subsequent epithelial degeneration.

In literature, no reports of cryopreservation of umbilical cord tissue have been found up to date, and, in most studies of tissue cryopreservation, the histological findings vary. Gaucher et al. (2011) observed the presence of vacuolated cells and a decrease in proliferative activity of keratinocytes in skin tissue after slow cooling. Kim et al. (2011) observed the presence of vacuoles and mitochondrial deformations in ovarian tissue after vitrification. Thomaz et al. (2005) noticed the presence of hyaline degeneration, cytoplasmic vacuolation, pyknosis and stromal lysis in rabbit oocytes after slow cooling process. This difference between the results of the literature and data found in this study can probably be attributed to the fact that the umbilical cord is a mucosal tissue, and, as in all mesenchymal tissue, edema is the most easily found reaction after any stressful situation on the tissue, being not a specific but a general feedback with no possibility of correlation with etiopathogenesis in particular cases (Filho 2011). Besides the studies that found histological changes, there are also studies in the literature that reported that cryopreservation did not induce any morphological changes, such as the vitrification of ovarian tissue (Keros et al. 2009), and the slow cooling of testicular tissue (Kvist et al. 2006). This heterogeneity of data about morphological changes after cryopreservation may be due, beyond the differences between the tissue, to the variety of protocols used for cryopreservation.

In addition to the morphological changes in this study, a significant reduction was observed in cell viability of umbilical cord tissue after thawing when compared with their respective fresh samples. After slow cooling, a significant reduction of cell viability was observed for both Wharton’s jelly and cord lining membrane samples. After vitrification, viable cells were not detected in any Wharton’s jelly sample and neither in most of the cord lining membrane samples (9/10). Possibly, there are three main causes for the loss of cell viability after slow freezing and vitrification. The first can be attributed to the cytotoxicity caused by cryoprotectants. According to Wang et al. (2007), cytotoxicity caused by cryoprotectants can be directly proportional to the concentration and to the temperature at which the samples are exposed. In this study, were used cryoprotectant solutions cooled down at 4 °C, which, in theory, reduces the cytotoxicity. However, very high concentrations of cryoprotectants were used, leading to increased cytotoxicity and, probably, being the main cause of cell viability reduction. Secondly, direct exposure of the tissue to liquid nitrogen may have caused cell damage and contributed to the decrease of cell viability after vitrification. It is likely that if the fragments were accommodated in straws before the contact with the liquid nitrogen, as done in other studies (Huang et al. 2005; Keros et al. 2009; Curaba et al. 2011), the damage could have been smaller. Finally, another possibility for loss of cellular viability after cryopreservation would be the occurrence of osmotic shock caused by thawing strategy used, in which the samples were removed from cryoprotectant solutions and sent directly to base medium. Studies show that thawing by multiple steps may be more efficient. Kvist et al. (2006) used a three step strategy to thaw the samples: after heating in warm water, the tissue were left for 5 min in solution containing ethylene glycol and sucrose, followed by 5 min in sucrose solution and, finally for 5 min in PBS without cryoprotectants, achieving good results in cell viability. In another study, Keros et al. (2009) used a four solution strategy for sample recovery after vitrification. The first consisting of propanediol and sucrose, the second being the same composition of the first but with a lower concentration of sucrose, the third composed only of sucrose, and the last consisting of only Cryo-PBS, and also obtaining good cell recovery. Perhaps if the thawing had been performed in multiple steps, with the presence of cryoprotectants in first solutions, the cell preservation could have been higher.

It is possible to notice that the decrease in cell viability was not related to the presence of morphological changes detected, since the samples with higher degree of interstitial edema did not necessarily showed lower cell viability. Probably the interstitial edema found did not affect survival or cell viability, as the intracellular edema (or vacuole) does. Kim et al. (2011) suggested that the presence of vacuoles may have affected the cell viability. In the literature, studies have also detected a significant decrease in cell viability after cryopreservation in different evaluated tissue. Succu et al. (2008) showed significant loss of cell viability in sheep oocytes after vitrification. Kim et al. (2011) observed that both slow cooling and vitrification processes induced reduction in cell viability in murine ovarian tissue. Moreover, Salvetti et al. (2010) reported that vitrification had worse results for cell viability than slow cooling in rabbit oocytes.

Although cryopreservation protocols used have caused cell viability reduction, the ability of cell proliferation in culture was preserved in most of the samples. After slow cooling, most of the cord lining membrane samples (9/10) and Wharton’s jelly samples (8/10) showed culture growth, in contrast to vitrification, in which most of the samples were not successful in cell culture. Considering the use of propanediol, a permeable cryoprotectant highly toxic to the cells, and the direct contact with liquid nitrogen in vitrification, these could be explanations for the lower efficiency of vitrification in preserving the tissue morphology, cell viability and proliferation capacity in vitro. In fact, Oskam et al. (2011) reported that the propanediol showed irreversible deleterious effects on sheep ovarian tissue, particularly follicular loss, and that some studies with the same tissue even achieved the recovery of cell viability, but none of them managed to recovery of gonadal function after transplantation (Newton et al. 1996; Gook et al. 2001).

However, 3/10 samples of the cord lining membrane grew in cell culture after vitrification, revealing that, for some samples, there are cells with proliferative capacity after vitrification process. The degeneration of the cord lining membrane epithelial, also detected in these samples, did not affect the proliferative capacity of cells in culture since fibroblasts, which are present in the subamniotic region of the connective tissue (Kita et al. 2010), are the target cells of the cell culture performed in this study. Despite the growth in culture, only one of the three samples showed viable cells by Trypan blue method. This result may be reflecting a limitation of the Trypan blue method, which indicates which cells do or do not have changes in the plasma membrane, which allow dyes to penetrate into the cells, but does not reflect the proliferative or functional capacity of cells (Gouk et al. 2011). Although this method is limited to the assessment of cell viability, the results showed that the vitrification protocol used was not suitable for the cryopreservation of umbilical cord tissue, since most samples were not successful in cell growth in vitro.

The time in which the samples were frozen (at −196 °C) ranged from 5 to 78 days (Table 1). The results indicate that this does not seem to interfere with the quality of the samples for growing in culture after freezing, since for both samples with short and long freezing period, the results were similar regarding histological data, cell viability and cell culture. Most published studies have also reported a short period of storage of cryopreserved samples, ranging from 1 week (Goud et al. 2000) to about 5 months (Lehle et al. 2005; Yu-Bin et al. 2007; Yu et al. 2007; Gonda et al. 2008). It has been well-established that the storage time in liquid nitrogen does not affect the viability of the samples after freezing, since at the temperature of −196 °C the cellular metabolism is completely stopped, effectively preventing all chemical reactions driven by heat (Mazur 1984). Thus, the factors that actually interfere with cryopreservation success are the processes of freezing and thawing (Karlsson and Toner 1996).

As for the cytogenetic results, no fresh sample had chromosomal abnormality, while after the process of cryopreservation, only two cultures showed tetraploid karyotypes: a Wharton’s jelly sample post-slow cooling and a cord lining membrane post-vitrification (Table 1). Previous studies showed that slow cooling induces polyploidy in mice and humans oocytes (Glenister et al. 1987; Al-Hasani et al. 1987; Bouquet et al. 1992), in addition, it increases the rate of chromosome misalignment in the metaphase II (Coticchio et al. 2009). However, it is hard to state that the cytogenetic changes were caused as a result of cryopreservation, since only two cultures showed chromosomal abnormalities. Still, it must be taken into consideration that the very long-term culture can induce the appearance of tetraploidy, and the longer the time in cell culture, the greater the possibility that changes may occur (Benkhalifa et al. 1993). However, in this study, the period of cell culture seems to have no influence on the tetraploidy occurrence, since both tetraploid samples remained in culture for approximate the same period of the respective group means. The time it remained frozen does not seem to have contributed to the tetraploidy occurrence, since for both samples the period was shorter than the others (Table 1). The tetraploidy may still have arisen as a result of cell fusion induced by DMSO, even DMSO also causes the membrane to become floppier, which would enhance permeability, facilitate membrane fusion, and enable the cell membrane to accommodate osmotic and mechanical stresses during cryopreservation (Notman et al. 2006).

Overall, the results of this study showed that the slow cooling protocol used was more efficient than the vitrification for cryopreservation of umbilical cord tissue. The success of slow cooling for other tissue and cell types is well-documented in the literature, with several studies showing good results (Donnez et al. 2004; Oktay et al. 2004; Meirow et al. 2005; Kvist et al. 2006; Gonda et al. 2008). It has also been reported that vitrification was not effective, like Succu et al. (2007) who reported that vitrification causes molecular changes that affect the development of various cells features (Succu et al. 2007). However, other studies have shown success in achieving vitrification (Yokota et al. 2000; Huang et al. 2008; Yu-bin et al. 2007; Keros et al. 2009; Gouk et al. 2011). Some of these studies used thin straw to accommodate the small tissue fragments and then plunged them into liquid nitrogen (Huang et al. 2005; Keros et al. 2009; Curaba et al. 2011). Yu-bin et al. (2007) described a method based on sending the tissue directly into liquid nitrogen and they obtained a successful cryopreservation of ovarian tissue. Considering this is an easy and cheap method, we decided to test it. However, we have not succeeded in the cryopreservation of umbilical cord tissue with this protocol. The use of the straws for the vitrification process may be an alternative to improve the obtained results.

This heterogeneity results in the literature, is due to different factors that can influence the process of cryopreservation. As for slow cooling and to vitrification, there are variety of protocols, using different cryoprotectants at different concentrations, temperatures and time, and other variables related to the thawing process. Still, it is necessary to consider the differences between tissue and studied cell types, since they all have their own biological features and the same cryopreservation protocol used successfully for a tissue may not be effective for another. As occurred in this study, in which the vitrification protocol was used successfully by Yu-bin et al. (2007) for ovarian tissue, predominantly epithelial, but for umbilical cord tissue, composed primarily of mucosal tissue was not efficient.

In conclusion, this study showed that it is possible to cryopreserve tissue fragments from the umbilical cord and, after thawing, to obtain viable cells capable of proliferation in vitro, contributing to the creation of a bank of frozen tissue, which may offer possibilities of using this tissue for a varied of purposes. The slow cooling protocol used was more efficient than the vitrification for cryopreservation of umbilical cord tissue, but more studies with larger number of samples, testing other cryoprotectants, and assessing other parameters such as, ability of preserving stem cells from umbilical cord tissue, should be conducted to enhance the results here obtained.

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© Springer Science+Business Media B.V. 2012