Biopolymer gels as a basis of cryoprotective medium

for testicular tissue of rats

Nataliia Volkova

Mariia Yukhta

Anatoliy Goltsev

Received: 10 September 2018 / Accepted: 17 November 2018

Ó Springer Nature B.V. 2018

Abstract Cryopreservation of testis tissue is a

promising approach to save fertility in prepubertal

boys under going gonadotoxic cancer therapies. The

using biopolymers as a basis of cryoprotective

medium can be effective for the optimization of

cryopreservation protocols of immature testicular

tissue. The research purpose was to determine mor-

phological parameters and metabolic activity of

seminiferous tubules of immature rat testes under

exposure to cryoprotective solution (DMSO) based on

collagen or ﬁbrin gels (CG or FG) as one of the ﬁrst

stages of developing the cryopreservation protocol. It

was found that 30-min exposure of tissue samples to

CG and FG with 0.6 M DMSO did not impair the

spermatogenic epithelium and metabolic activity of

the cells (MTT test and total lactate dehydrogenase

activity). The use of FG at the time of exposure of

45 min did not lead to signiﬁcant changes in the

metabolic activity in contrast to other groups. The

ﬁndings could be used to substantiate and develop the

effective techniques for cryopreservation of immature

seminiferous tubules.

Keywords Immature testicular tissue 

Spermatogenic epitelium  Cryoprotective medium 

Collagen gel  Fibrin gel  Metabolic activity

Introduction

In prepubertal boys affected by cancer, cryopreserva-

tion of testicular fragments prior to gonadotoxic

treatment is an ethically accepted strategy to save

fertility (Abrishami et al. 2010; Michele et al. 2017).

Frozen immature testicular tissue can later be used for

completion of spermatogenesis after in vitro matura-

tion, germ cell transplantation or testicular tissue

grafting (Travers et al. 2011). Despite the fact that the

low temperature preservation of testis is associa ted

with some difﬁculties (different cell size, post thawing

ischemia), it can allow to preserve the different

testicular cells in their ‘‘niche’’ with respect interac-

tions between germ cells and Sertoli cells (Milazzo

et al. 2010). Nevertheless, the optimization of cryop-

reservation protocols for improving recovery has not

been practically investigated. In our opinion, the

various biotechnological methods using natural

biopolymers can be effective for this purpose, but

they require additional studies.

Tissue fragment encapsulation is based on cell

scaffold technology (Nicodemus and Bryant 2008).

The latter relies on immobilization of cells in a

N. Volkova (&)  M. Yukhta  A. Goltsev

Department of Cryopathophysiology and Immunology,

Institute for Problems of Cryobiology and Cryomedicine

of the National Academy of Sciences of Ukraine, Kharkiv,

Ukraine

e-mail: [email protected]

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https://doi.org/10.1007/s10561-018-9740-z

(0123456789().,-volV)(0123456789().,-volV)

biomaterial which allows bidirectional diffusion of

nutrients, oxygen, and waste, thus promoting cell

interactions. Substances for encapsulation should also

be biocompatible and favor (subject to application)

vascular invasion and tissue integration in the host

(Dhandayuthapani et al. 2011; Jafari et al. 2017).

Polymers are very often used as scaffolds. They can

be synthetic or biological origin, and degradable or

non-degradable. The two main classes of natural

biopolymer are proteins and polysaccharides (Guarino

and Ambrosio 2014; Jafari et al. 2017).

Collagens are most abundant family of the complex

enzymatically degradable proteins that has unique

biological properties . They are involved in forming of

membranes, ﬁbrillar system and other components of

the extracellu lar matrix, dete rmining structural integ-

rity and physiological functions in cells (Gelse et al.

2003). They have been oftentimes applicated for

biomedical investigation (Nair and Laurencin 2007;

Bret et al. 2011). The studies in ﬁeld of cryobiology,

conducted by Allenspach and Kraemer (1989) showed

that the distribution of water and structure of ice

crystals in collagen gels depend on the composition of

the gel and the program of freezing. Thus, the presence

of an extracellular matrix may affect the structure of

ice formation during freezing-warming, and hence the

ﬁnal result of cryopreservation.

Now, using of ﬁbrin gel is widespread as an

alternative to collagens. The products derived from

autologous blood are used in the regenerative

medicine for the recovery of damaged tissues and

organs. One of the important properties of ﬁbrin gel is

ability to degrade in a controllable method and self-

organizations into a polymer system that imitates of

blood coagulation. At the same time, ﬁbrin gel and

other derivatives of blood (serum, plasma) contain a

large number of different bioactive substances that

used as protectors from the damages during the

cryopreservation of the cells (Nair and Laurencin

2007; Li et al. 2015).

The apply of gels as a matrix for fragments of the

tissue of testicles will allow to minimize tissue damage

during cryopreservation by reducing the amount of

free water and increasing of tissue resistance to

overcooling. In addition, it is known that biopolymer

gels have high viscosity which makes them capable to

inﬂuence the proce sses of ice crystal formation

inhibiting their growth in volume and thus reduce

the degree of mechanical action on the tissues (Koebe

et al. 1990; Takahashi et al. 1988).

In our preliminary study, it has been shown that a

30-min incubation of samples of tubules of immature

rat testes in Hanks medium with add of 5% BSA and

0.6 M DMSO did not cause change to the morpho-

logical characteri stics of tissue and metabo lic param-

eters of cells (Volkova et al. 2017). The aim of this

research was to determine morphological and func-

tional parameters of seminife rous tubules of immature

rat testes under exposure to cryoprotective solu tion

(DMSO) based on collagen and ﬁbrin gels as one of

the stages in optimizing of the cryopreservation

protocol.

Materials and methods

Animals

Outbreed white sexually immature male rats

(50 ± 15 g weight, aged 7–8 weeks, n = 50) were

used in the study. All the manipulations were carried

out in accordance to the European convention for the

protection of vertebrate animals used for experimental

and other scientiﬁc purposes (Strasbourg,

18.III.1986). The protocols were approved by the

Bioethics Committee of Institute for Problems of

Cryobiology and Cryomedicine of the NAS of Ukraine

(Permit No 2016-05).

Preperation of biopolymer gels

Collagen gel (CG) was prepared using collagen type I,

which was obtained from rat tendons as described by

Chandrakasan et al. (1976). The pH was brought to

neutral using 0.34 N NaOH solution.

Fibrin gel (FG) was received from the fresh

autologous blood of animals, which was obtained

from a cardiac vein and centrifuged for 12 min at a

rate of 1000 g. After centrifugation, three fractions of

blood were received: the lower fraction was erythro-

cyte mass; the upper fraction was platelet-poor

plasma, and the medium one was platel et-rich ﬁbrin

gel. Only blood samples without signs of hemolysis

were used in the work.

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Cryoprotective media

We used DMSO (PanEco, Russia) as the widespread

cryoprotectant for testicular tissue freezing at ﬁnal

concentrations of 0.6 M (Keros et al. 2007), but

cryoprotective media differed in the basis. The ﬁrst

experimental medium (CG ? DMSO) consisted of

collagen gel with 0.6 M DMSO, and the second one

(FG ? DMSO) consisted of ﬁbrin gel with

0.6 M DMSO. The controls were corresponding gels

without cryoprotectant (CG and FG respectively) and

intact samples of testicular tissue.

Exposure of testicular tissue

Samples of testicular tissue weighing 75 ± 5 mg were

isolated mechanically immediately before the study.

They were immersed in the cryoprotective media and

exposed during 15, 30, 45 and 60 min at 4 °C (Keros

et al. 2007; Milazzo et al. 2010; Travers et al. 2011).

The samples after exposure were (washe d out by a

three-step change of Hanks solution and used for

morphological and biochemical studies (Fig. 1).

MTT test

Quantiﬁcation of metabolic activity was executed at

15, 30, 45 and 60 min of term expositions on testicular

tissue samples. MTT test is based on the ability of

dehydrogenases to restore [3-(-4,5-dimethylthiazolyl-

2)-2,5-diphenyltetrazolium bromide] in living cells to

blue crystals of formazan, which are insoluble in

water. A ﬁnal concentration of 0.5 mg/mL MTT

(Fluka, Germany) was added to the samples and

incubated for 3 h at 37 °C. Then the medium was

deleted and 100% DMSO was added to each samples

of tissue to solubilize the precipitate (Mossman 1983).

Absorbance was read at 570 nm.

Analysis of total lactate dehydrogenase (LDH)

activity

The total LDH activity was estimated quantitatively

by the method of UV spectrophotometry was using

test kits (Felicit, Ukraine). The samples of tissues of all

experimental groups were homogenized and ﬁltered,

with following centrifugation (1000 g for 10 min).

Reaction was started by the addition of enzyme sample

to 20 lL supernat ant of tissue homogenat with

following spectrophotometrical measuring of a

decrease in absorbance of NADH at 365 nm.

Histomorphometry

Histomorphometry was carried out maintaining blind-

ing by involving the third person who did not take part

in the experiment. Parafﬁn blocks were prepared and

slices of 7 lm thickness were stained by hematoxylin

and eosin. Histological preparations were studied

using Axio Observer Z1 inverted microscope (Carl

Zeiss, Germany). Obtained image ﬁles were processed

using the Axiovision v. 4.8 (Carl Zeiss).

The following qualitative criteria were studied: cell

retraction, nuclei condensation, cell detachment and

formation of gaps (Fig. 2). Additionally, a cell density

Fig. 1 Experimental scheme. CG collagen gel, FG ﬁbrin gel,

DMSO dimethyl sulfoxide, MTT 3-(4,5-dimethylthi azol-2-yl)-

2,5-diphenyltetrazolium bromide, LDG lactate dehydrogenase

Fig. 2 Histological evaluation of spermatogenic epithelium

damages. R cell retraction, N nuclei condensation, D cell

detachment, G formation of gaps

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of spermatogenic epithelium was detected as the

average of the numb er of nuclei in the section area

(0.102 mm

) with following conversion per 1 mm

Statistical analysis

The Mann–Whitney U-criterion was used to determine

the statistical signiﬁcance of the differences in

continuous variables when comparing between the

groups with multiple (more than two) comparisons

Kruskal–Wallis ANOVA tests using Excel (Microsoft,

USA) and Statistics 8 (StatSoft, USA) software.

Results

Metabolic activity

The obtained experimental dates of the MTT test

indicate that the 15- or 30-min exposures in all studied

media did not lead to a signiﬁcant change in metabolic

activity of the immature testis tissue if compared to the

intact control and between the investigated biopoly-

mers (Fig. 3). In addition, the metabolic activity was

decreased in groups with CG by 19.5% and CG ?

DMSO by 21.9% compared to intact control at the

45 min. In the media based on FG this parameter

remained at the level of intact control. After 60 min of

exposure the investigated index in all cases was lower

if compared to intact control: for CG and CG ?

DMSO by 37.5 and 32.7%, for FG and FG ? DMSO

by 17.3 and 19.2% respectively.

The results of the study of the inﬂuence of

cryoprotective media on the total LDH activity are

shown in Fig. 4. The time of exposu re of 15 and 30

min in all tested biopolymers media did not lead to

strong changes in LDH activity if compared to the

intact control as well as between the investigated

media. At the 45-min exposure there was a tendency to

decrease the investigated index in the CG and

CG ? DMSO media by 17.2 and 18.3% respectively

compared to the intact control. At 60-min exposure of

tissue in media based on CG, we observed a decrease

in LDH act ivity index by more than 1.5 times if

compared to intact control. Thus, the increase in the

tissue exposure time in the media on base of CG more

than 30 min resulted in signiﬁcant changes in LDH

activity, which probably indicated the violation of

metabolic processes in the cells. The use of FG at the

timing of exposure of 15–45 min did not lead to

signiﬁcant changes in this parameter. The data of the

LDH activity on the 60th minute of observation are

consistent with the results of MTT test at the same

time, which indicates the inhibition of metabolic

processes in experimental samples due to the exposure

duration.

Histomorphometry

Dates of the biochemical study showed no inﬂuence of

investigated biopolymers on seminiferous tubules of

testes under the 30-min term of exposure. Signiﬁcant

changes were noted starting from 45 min. Therefore

the next step was histomorphometric analysis to obtain

Fig. 3 Dynamic of metabolic activity (MTT test) of seminif-

erous tubules of immature rat testes under exposure to

investigated media. Solid line is an index of intact control.

*The difference is statistically signiﬁcant relative to intact

control (M ± m; n = 5; p \ 0.05)

Fig. 4 Dynamics of total LDH activity of seminiferous tubules

of immature rat testes under exposure to investigated media.

Solid line is an index of intact control. *The difference is

statistically signiﬁcant relative to intact control (M ± m; n = 5;

p \ 0.05)

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more information about structural characteristics at

this term of exposure (Fig. 5).

It was found that seminiferous tubules of the intact

group had a form of the rounded formations of the

basal membrane inside of which there was a multilayer

germinative epithelium (Fig. 5 Intact control). The

population of spermatogenic epithelium was repre-

sented by all types of spermatogenic cells excepted of

spermatozoa. Spermatogonia were located on the

basal membrane, above them, spermatocytes were

placed in 2–3 rows. In some tubules, the cells reached

a developmental level of early spermatids.

After 30-min exposure in CG (Fig. 5 CG), in the

most case s, there was slight desquamation of epithe-

lium into the lumen of seminiferous tubules. The

detachment of sperma togenic epithelium from the

basal membrane was partial. In addition, there was a

chaotic location of the germinative cells with forma-

tion of gaps and ruptures inside the spermatogenic

epithelium due to the obvious cell retraction. Nuclei

condensation was slightly present.

In the case of CG ? DMSO combination, germi-

native cells in most cases saved their connection with

the basal membrane and orientation within the sper-

matogenic epithelium despite the obvious retraction

and gap formation. However, there were extensive

regions covered by karyolysis (the cell nuclei are

swollen, slightly colored with hematoxylin, without

distinct contours) in the center of separate seminifer-

ous tubules. At the same time, the tubules were

detected in which spermatocytes had a hyperchromic

nucleus and a sharp eosinophilic cytoplasm (Fig. 5

CG ? DMSO).

When FG was used, cell nucl ei of the primarily

spermatocytes had a swollen appearance and were

sharply hyperchromic. The contours of seminiferous

tubules were fuzzy and deformed but desquamation

phenomena were absent. The cytoplasm of germina-

tive cells was slightly retracted (Fig. 5 FG).

After exposure in FG ? DMSO, as well as in the

previous case, cell nuclei of the spermatogenic

epithelium in some seminiferous tubules had a swollen

appearance and were sharply hyperchromic. But it

should be noted that the FG, in contrast to the CG, had

a more pronounced protective effect in combination

with DMSO, preventing the development of necrosis

in germinative cells (nuclei condensation was slightly

present). Cell retraction and formation of gaps in most

cases were slightly present too and cell detachment

was absent (Fig. 5 FG ? DMSO).

The morphometric study showed that signiﬁcant

differences in the average density of spermatogenic

Fig. 5 Seminiferous tubules of intact immature rat testes

(a) and after 30-min exposure in the CG (b), CG ? DMSO

(c), FG (d), FG ? DMSO (e). Hematoxylin and eosin staining.

R cell retraction, N nuclei condensation, D cell detachment,

G formation of gaps, 1 regions covered by karyolysis, 2 swollen

and hyperchromic nuclei

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cells were absent in both investigated media without

cryoprotectant compared to the intact control (Fig. 6).

In the case of exposure of testicular tissue to DMSO

based on CG this parameter was in 1.3 times lower

than in the intact. However, this statement should be

treated with caution since according to the data

mentioned above some of the cells could have signs

of karyopicnosis/kariolysis. The use of FG as a base of

medium for cryopres ervation was more effective in

contrast to CG. In this case (FG ? DMSO), the

average cell density did not differ signiﬁcantly from to

the intact control.

Discussion

Now it is absolutely clear that progress in the ﬁeld of

biotechnology is inextricably linked with the devel-

opment of cryobiological approaches because low

temperature preservation allows storing biomaterial

for future medical procedures. Furthermore, cryop-

reservation makes the use of cell and tissue products

easy in time and place. Therefore, materials for such

purposes should be acceptable and even effective in

the process of freezing–thawing. This work is the ﬁrst

stage in the study of the possibility to use the

biopolymer gels for improving of the efﬁciency of

immature testicular tissue cryopreservation that is so

necessary for prepubertal boys undergoing gonado-

toxic therapy.

Modern development of innovative biomaterials

has made a signiﬁcant contribution to the development

of reproductive medicine, especially in the aspect of

maintaining fertility. At the moment hydrogels are

considered as a unique solution for stem cell storage

and transport. Their effectiveness has been studied in

relation to embryonic stem cells (Ji et al. 2004),

multipotent stem cells from bone marrow (Chen et al.

2013), adipose tissue (Swioklo et al. 2016) etc.

Nevertheless, the using of gels for tissue cryopreser-

vation is insufﬁciently studied. Some recent studies

(Hatte et al. 2016) have shown that DMSO with

polysaccharide-based hydrogel can be recommend ed

as a new effective tool for improving procedure of cell

or tissue low temperature preservation. The authors

note that this combination allow to ensure safety

discarding serum and to reduce DMSO concentration

and its cytotoxicity or clinical side-effects. Other

results (Itoh et al. 2001) indicate that covering with

CG improved the recovery and viability of the small

preantral fol licles during vitriﬁcation steps. Further-

more, the vitriﬁed follicles embedded in CG could be

maintained in culture during 1 week. Microencapsu-

lation in ultrahigh viscous alginate allows maintaining

of neurosphere morphology, membrane integrity of

cells, their metabolic activity, and cell-speciﬁc inter-

actions, which are major requirements for their

applications after thawing. The use of gels in cryop-

reservation has also been studied in relation to

hepatocyte spheroids (Lee et al. 2004) and multicel-

lular tumor spheroids (Sakai et al. 2012) and opti-

mistic results were received. In the same way FG

provides optimal support for adhesion, proliferation,

differentiation and biochemical signaling of cells. It is

used to encapsulate fragments of integral tissue or

isolated cells to support their 3D structure, as well as to

study biological phenomena that would be impossible

in 2D systems (Chiti et al. 2017). The studies,

conducted by Murdock et al. (2018) showed that

extracellular matrix can promotes mitogenesis, migra-

tion, and/or differentiation of various stem/progenitor

cells and contribute to the survival of human sper-

matogonial stem cells in culture.

So encapsulation is considered at this point as a

promising approach for low temperature preservation

of 3D cell systems, as this approach protected cells

against mechani cal damages during ice crystal forma-

tion and reduce the risk of cell–cell disruption through

immobilization within the hydrogel (Malpique et al.

2010).

In this research, we examined the potency of the

using of collagen and ﬁbrin gels as the basis of the

cryoprotective medium at the exposure stage. Colla-

gen and ﬁbrin gels are commonly used in tissue

Fig. 6 The average density of spermatogenic epithelium cells

after 30-min exposure in the investigated media. The solid line is

an index of intact control. *The difference is statistically

signiﬁcant relative to intact control (M ± m; n = 5; p \ 0.05)

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engineering as the foundational matrix due to their

availability, scaffolding function, and bioactive qual-

ities. Undoubted advantage of them is an ability to

closely mimic native extracellular matrix of tissues.

Scaffolds on basis of this substance in various forms

have been studied and employed for different tissue

regeneration purposes (Geckil et al. 2010).

It was found that FG led to the prolongation of the

exposure time of the seminiferous tubules to 4 5 min

according to the results of metabolic tests as well as to

less histological changes in the tubular structure with

preservation of the average cell density of the

spermatogenic epithelium. More pronounced protec-

tive effect of FG in our opinion is due to the presence

of different biologically active substances in its

composition in contrast to CG, whose composition is

poorer. It is known that various blood products contain

a number of growth factors (hepatocyte growth factor

(HGF), transforming growth factor b (TGF-b), plate-

let-derived growth factor (PDGF), insulin like growth

factor 1 (IGF-1), vascular endothelial growth factor

(VEGF), epidermal growth factor (EGF), nerve

growth factor (NGF) etc.) but in different ratio

(Nishiyama et al. 2016). Growth factors play a huge

role in the cell functioning and can affect cell

metabolic processes including on the stages of low

temperature preservation. Thus a signiﬁcant improve-

ment was observed in viability, motility and apoptosis

level of human asthenozoospermic sperm after addi-

tion of NGF to the cryoprotective media (Saeednia

et al. 2016). The using of IGF-I reduced the ratio of

sperm with disrupted membranes and the number of

Annexin V? sperm after hypothermic storage

(Makarevich et al. 2014). VEGF treatment can prevent

germ cell death in bovine testis tissue explants due to

stimulation of an intracellular response (Caires et al.

2009). Moreover in the case of application of cryop-

reserved testicular tissue in vivo VEGF can support

engraftment of the transplant promoting angiogenesis

in the tissue (Tian et al. 2016; Del Vento et al. 2018).

Thus the use of FG as an integr al component of

freezing medium is effective and contributes to the

preservation of the average density of germinative cell

and their metabolic activity during the exposure stage

of cryopreservation. We recommend an optimal

exposure time of 30-min in the case of cryoprotective

media on the bases of CG or FG. However, 45-min

exposure of testicular tissue to FG with 0.6 M DMSO

did not impair the spermatogenic epithelium and the

metabolic activity of the cells (MTT test and LDH

activity) and therefore time interval can be increased

for this gel. In the future, we plan to study the

morphological and functional parameters of immature

testicular tissue after freezing- thawing and evaluate

the contribution of biopolymer gels to the protection of

the spermatogenic epithelium during low-temperature

storage.

Funding This study was carried-out within the research

project of the National Academy of Sciences of Ukraine (No.

2.2.6.99).

Compliance with ethical standards

Conﬂict of interest All authors declare that they have no

conﬂict of interest.

Ethical approval All the manipulations with animals were

carried out in accordance to the European convention for the

protection of vertebrate animals used for experimental and other

scientiﬁc purposes (Strasbourg, 18.III.1986). The protocols

were approved by the Bioethics Committee of Institute for

Problems of Cryobiology and Cryomedicine of the National

Academy of Sciences of Ukraine (Permit No 2016-05).

Human and animal rights This article does not contain any

studies with human participants performed by any of the

authors.

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