Recent advances in developing 3D culture systems of spermatogonial stem cell preservation and differentiation: A narrative review

Abstract Male infertility has received vast attention in recent years and has no clear etiology in almost 40% of cases. Several methods have been suggested for preserving sperm and spermatogonial stem cells (SSCs) in both in vivo and in vitro conditions. The efficacy of these methods is related to their abilities, including providing an optimal environment for sperm preservation and long-term SSC culture for in vivo and in vitro differentiation of these cells. In this review article, a full MEDLINE/PubMed search was performed using the following search terms: “Spermatogonial Progenitor Cells, Stem Cells, Fertility Preservations, Sperm Freezing, Cell Differentiations, Tissue Scaffold, 3-Dimensional Cell Culture", which retrieved results from 1973-2022. Related articles were added to the bibliography of selected articles. Exclusion criteria included non-English language, abstract only, and unrelated articles. The production of functioning male germ cells is suggested by introducing modern bioengineered systems as a new hope for the maintenance of male fertility. Till now, few in vitro spermatogenesis investigations have provided appreciable amounts of mature gametes. Each method had benefits and disadvantages, but the 3-dimensional culture method had the greatest impact on the differentiation and preservation of SSCs. One of the critical elements of research is the preservation of sperm and the differentiation of SSCs. Several methods have been employed in this area. Various scaffolds providing an environment similar to an extracellular matrix and conditions for germ cell development and survival have been employed in recent research.


Introduction
Fertility in both men and women has recently attracted attention.Due to continuous low total fertility rates below replacement level (2.1 children per woman), several countries in Asia and the European Union are expected to soon see considerable population declines (1).Some studies revealed a significant decline in semen quality during the past 50 yr (2, 3).Around 40-50% of infertility cases are caused by the "male factor", and up to 2% of males worldwide have sperm with poor quality.As a result, maintaining male fertility is crucial (4).Several disorders and diseases that impact male fertility are more prevalent now than they previously were.The fertility preservation procedure might be required for some systemic diseases or medical therapies that could impair fertility (5).
Non-oncological factors, including genetic disorders, inflammation induced by trauma, infections, and autoimmune diseases, are considered risk factors for male infertility (6)(7)(8).Reduced sperm production and function; however, may result from the underlying illness (5).Serious trauma, such as spinal cord injuries linked to erectile and ejaculatory dysfunction, is on the opposite end of the non-oncological spectrum (9,10).Certain cancers that develop before or after puberty require preserving fertility issues because of chemotherapy and radiation treatments.80% of cancer survivors are considering starting a family, but only 10% would use donor sperm or adoption.
Vasectomy is one of the other conditions (11), up to 6% of men eventually ask for a reversal when their circumstances and objectives alter (12).In light of this, the patient might be advised about the possibility of sperm freezing before a vasectomy or sperm extraction from epididymis during surgery (13).
Postpubertal males who produce completely developed sperm have the opportunity to have their semen cryopreserved, which allows them to have children later in life through in vitro fertilization (14).Since young boys are unable to produce sperm until puberty, it will be difficult for them to maintain fertility (15).Postpubertal males can be offered a testicular biopsy which contains spermatogonial stem cells (SSCs) (16).SSCs are a subpopulation of type A spermatogonia localized in the seminiferous tubules' basement membrane, and they are essential to the process of spermatogenesis, which produces millions of haploid spermatozoa every day (17).Potentially, isolated SSC or immature testicular tissue can be developed in vitro or returned to the natural physiological microenvironment.Developing SSC niches would make it easier to create a successful long-term SSC culture in vitro, which is the basis for further study and applications (16).
The preservation and culture of germ cells are suggested methods for preserving fertility in these people.In this review, we will focus on the procedure for differentiating and maintaining germ cells and sperm both in vitro and in vivo.

Materials and Methods
The MEDLINE/PubMed database were thoroughly searched and 6489 articles were found.The following MeSH phrase combinations were used to search "Spermatogonial Progenitor Cells" AND "Stem Cell" AND "Fertility Preservations" AND "Sperm Freezing" AND "Cell Differentiations" AND "Tissue Scaffold" AND "3-Dimensional Cell Culture".

SSCs preservation and differentiation
The exclusion criteria were as: abstract only, non-English language, and unrelated articles.
Following the search, it was found that data

Orgin of SScs
In the postnatal testis, SSCs develop from gonocytes, which grow from primordial germ cells (PGCs) during fetal development (18).In

Cellular and molecular pathways in mammalian testis differentiation
The The maturation of B spermatogonia results in the production of primary and secondary spermatocytes, spermatids, and spermatozoa (29).
After adolescence, additional mitotic and meiotic divisions occur in the second division, which leads to produce primary and secondary spermatocytes and, eventually, spermatozoa.
Since 1960, several studies have focused on how to produce meiotic and postmeiotic alterations in male germ cells in vitro.For many years, a major area of study focused on the fundamental principles of male germ cell differentiation, which produces mature haploid spermatozoa inside structurally well-organized tissue.However, according to other investigations, spermatogenesis was stopped before it reached the meiotic phase (31).Another important characteristic of the testis is its dual functions as a glandular and gonad tissue, the in vitro production of sperm from male germline stem cells is still considered difficult because the entire spermatogenic process must occur in a cell culture plate.Also, differentiating SSCs' requires a varied set of paths.The molecular process is crucial for the maintenance and differentiation of testis reproductive cells, which should be considered in vitro culture (32).
Also, molecular pathways are crucial in these cells' differentiation ability.Retinoic acid (RA) and the RA receptor are both necessary for triggering meiosis (33).This stimulates differentiation and blocks factors like promyelocytic leukemia zinc finger (34).
The Sertoli cell-secreted stem cell factor can bind to the proto-oncogene receptor tyrosine kinase, which starts a signaling cascade for differentiation (35).The antagonist of BMP4, Noggin, prevents RA-induced production of c-Kit and RA-8 (Stra8)-stimulated gene expression, but BMP4 and RA signaling cooperate.Neurogenin 3, whose function is not yet understood, is an intrinsic differentiation element (36).Sertoli cells release glial cell-derived neurotrophic factor (GDNF), and in mice with aging SSCs, GDNF expression is found to be decreased.

Preservation and differentiation of SSCs
The most advanced treatments for preserving male germline or fertility that are now available include testicular tissue freezing, SSC transplantation, and in vitro spermatogenesis (41).The primary experimental tactics have

Testicular organ culture systems
Testicular organ cultures from newborn mice were used in the first study on in vitro spermatogenesis.The first attempts to develop a culture method for testicular tissues were made in 1920.The testicle pieces were preserved in a culture medium based on plasma clot.The plasma clotting method is a culture that may be used to examine the development of fetal organs and testicular tissue (47).The main benefit of this method was to enable the visual study of whole cells with an emphasis on their dynamic properties and shape modulations over time and in response to regularly controlled environmental changes (48).
Another technique is the raft one in which the organ was set on a lens made of paper or rayon to help transfer the tissue.Excess fluid was filtered out, and the net containing the tissue was repositioned on a new pool of medium.A size of around 8 mm is the largest that may be properly maintained.To prevent ripping or crushing of the tissues, great care should be used when dissecting these explants.This approach was utilized to research testis culture and cancer in in vivo models.The target tissue was placed on Trowell's grid system, which was made of perforated steel sheet that is hydrated up to the grid's surface, before being placed in a culture chamber.There is a challenge with the raft floating method.Skeletal tissues are frequently placed directly on the grid due to their higher degree of rigidity, whereas softer tissues, such as the skin or glands, must first be stretched out on rafts to be sustained throughout the grids.This is because different tissues have varying degrees of stiffness.The testis of the adult rat was organs grown using this method.However, the results revealed that most of the tubules had degenerated, with just a tiny number surviving for up to 3 days (49).
Based on a modified plasma clat approach, human epidermal cells were seeded on rafts made of collagen and fibroblasts, and an air-fluid was placed between the cells (50 Instead, Sertoli cells aggregated in the top layer. According to the immunocytochemistry data, Vimentin, a Sertoli cell marker, was found in aggregations (45).
A study revealed strong modeling of in vitro human testicular organogenesis from the fetal genital ridge, which represents a significant advance for the study of male reproduction.
Human gonads from aborted 12-19-wk fetuses were cut up and placed on agarose gel stands.
Additionally, the meiotic phases were completed, and the differentiation of spermatogonia was seen morphologically and in terms of gene expression (54). In

Hydrogels method
The extracellular matrix (ECM) mostly comprises proteins and polysaccharides.Laminin is a crucial modulator of protein, paracrine, autocrine growth factor, and Sertoli cell secretion and transfer in the basement membrane, which directly affect the survival and differentiation of testicular cells throughout development (59).
One of the fundamental signaling pathways, direct cell-to-cell contact, is crucial for the effectiveness of spermatogenesis and might be efficiently assisted by hydrogels (60).
Cells might be implanted in a thick layer Alginate can also lessen the toxicity brought on by freezing when used in cryopreservation.
Alginate hydrogels were tested in 2017 for their potential toxicity in the culture systems of SSCs.Surgery was used to extract the SSCs from immature mice that were 6 days old.SSCs were enclosed in alginate hydrogels.
The cells were removed from the hydrogel and analyzed for survival, cell shape and structure, cytotoxicity, and expression of the apoptotic genes following encapsulation and cultivation for a month in Dulbecco's Modified Eagle Medium culture media with 10 ng/ml GDNF.Compared to the control group, the main group's expression of the Fas Cell Surface Death Receptor (FAS) gene increased, whereas that of the Bax and P53 genes decreased.Cell viability was significantly reduced, but spermatogenesis was restored after the freeze-thawed SSCs encapsulated in alginate hydrogel.Various studies show that alginate hydrogels had high vitality (74.08%) and low cytotoxicity (5%), and protect SSCs from damage (62).

International Journal of Reproductive BioMedicine
Fath-Bayati et al.

Grafting method
Graft technic is an additional method for sperm storage that obtained full using tissue from all 3 species in 2002 by grafting tiny fresh biopsies from prepubertal mice, pigs, and goats onto the skin of nude mice.The study claims that the replacement of segments was just fixed subcutaneously without creating synthetic vascular anastomosis (74).
In the same nude mouse model, the

Preservation of fertility in adult males
For adult males and pubertal boys who produce sperm in the ejaculate and who will be receiving gonadotoxic treatment, the cryopreservation of ejaculated semen is advised.Gonadal shielding is a solution if sperm collection is impossible for males receiving radiation therapy (80).The procedure involves the collection of, preferably, 3 semen samples with an abstinence period of at least 48 hr in between samples, followed by the cryopreservation of the sperm samples (81).However, it is frequently necessary to collect more than one semen sample on the same day to prevent delay in oncological treatment.When ejaculation fails or there are no spermatozoa in the ejaculate, collection of the sperm from the epididymis through either percutaneous epididymal sperm aspiration or microsurgical epididymal sperm aspiration can be done (82).

Conclusion
Spermatogenesis is a susceptible process influenced by endocrine hormones and micro-environmental conditions.Several major illnesses might affect male fertility.These Therefore, tissue-engineered testis, can be helpful in achieving spermatogenesis for tissue transplantation or in vitro conditions.In the postpuberty period, sperm can be frozen and used for intracytoplasmic sperm injection under suitable conditions.Many hypotheses suggest that this maturation can be achieved through 2and 3D tissue culture, and sperm can be obtained.But using this system in different laboratories and providing setup brings various problems.
Standardized organoids and organoid scaffolds that can be developed for 3D cultures appear preferable.
In this review, the details of developing various techniques for both mentioned groups have been
the embryo's epiblast stage, which takes place around 7-7.25 days post coitum (DPC), a tiny group of alkaline phosphatase-positive cells is the first sign of PGCs, a temporary cell population.The extra embryonic ectoderm's expression of bone morphogenetic protein 4 (BMP4) and BMP8b is necessary for PGC specification, and BMP8b is from the extraembryonic ectoderm.When the allantois develops in mice between 8.5 and 12.5 DPC, PGCs start migrating from the hindgut to the indifferent gonad (19 2 main divisions of the mammalian testicular tissue are the seminiferous tubules and interstitial tissue.It has been demonstrated that the putative testis' tubular structures progressively divide into cords from an amorphous primordium.Seminiferous tubules in young men include various testis cell types and components, including SSCs, basement membrane (deposited by Sertoli cells), peritubular myoid cells, and Sertoli cells.However, in adult males, the SSCs undergo spermatogenesis to differentiate into mature spermatozoa.SSCs are responsible for maintaining the male germ cell pool and can self-renew (28).At the start of puberty, the Sertoli cells also go through major morphological and functional changes.At stages of development, these supporting cells generate certain types of proteins, cytokines, hormones, and growth factors that ultimately form the blood-testis barrier, playing a crucial role in spermatogenesis and testis formation.Like with all other germ cells, SSCs are in direct contact with Sertoli cells, SSCs and more mature germ cells cannot exist in vivo without Sertoli cells (29).Leydig International Journal of Reproductive BioMedicine SSCs preservation and differentiation cells, a distinct type of cell, are also found in the testicular interstitium and respond to luteinizing hormone by producing the steroid hormone (testosterone).This mechanism maintains postpubertal spermatogenesis and begins the androgenic consequences in the male fetus (30).In primates, there are 2 distinct spermatogonial subtypes.The A pale spermatogonia serves as progenitor cells that can divide to create both A pale and differentiated B spermatogonia, while the A dark spermatogonia are thought to be the testicular stem cell with strong proliferative activity.Incomplete mitosis and further differentiation in spermatogonia result in intermediate and B spermatogonia with cytoplasmic bridges.
been emphasized: SSCs transplantation or testicular tissue grafting into host animals and the differentiation of in vitro spermatogenesis.In vitro investigations have focused on preserving germ-cells and producing mature and functional spermatozoa.2 methods have been suggested for the maintenance of SSCs.Generating viable male germ cells is a new hope for maintaining male fertility that has been made possible by modern bioengineered systems.Future chances for genome preservation and fertility therapy for cancer patients of adult, juvenile, and prepubertal ages, as well as noncancerous infertile males, may be made possible by these procedures (42).Organ culture systems are considered valuable methods for studying pathophysiological processes because they can effectively represent the functions of the organ in a variety of conditions and environments.The lack of the typical tissue microenvironment can change cellular response (43).The essential cell-cell and cell-matrix signals involved in spermatogenesis are supported by 3D-techniques, which provide International Journal of Reproductive BioMedicine SSCs preservation and differentiationall the necessary information about this organ for in vitro spermatogenesis or the maintenance of male fertility(44).By providing better mimics of the species-and age-specific structures of the testis, as well as the biomechanical properties, cell-cell interactions, and natural matrix of the mammalian reproductive tract, 3D cultures that place cells on synthetic matrices, organoids, or organs-on-a-chip cultures, may be able to overcome the limitations of in vitro 2D culture vessels(45).Furthermore, organ cultures allow researchers to control the paracrine environment and separately evaluate the effects of each growth factor on the spermatogenesis process.Many techniques have been developed for the in vitro maintenance and culture of intact tissues.The contact between the seminiferous tubules and the interstitial area may be maintained in the 3D testicular tissue culture systems, which are suitable for the progression of spermatogenesis.To encourage in vitro spermatogenesis; however, such media must be improved by including specific necessary and efficient elements (such as luteinizing hormone, follicle-stimulating hormone, RA, triiodothyronine, testosterone, or other types of antioxidants, vitamins, growth factors, and hormones)(46).
using collagen gel matrices, creating an extracellular environment that successfully mimics the activities of the seminiferous tubule basement membrane.Using 2 classes of natural-based polymers, including proteins (like collagen) and polysaccharides (like chitosan and alginate), testicular cells have been ordered spatially in several investigations.The survival of testicular cells is improved by including laminas in the structure of collagen-based gels.In addition to these advantages, such 3D culture microenvironments prevent cell ischemia, particularly in the long run (61).Some of the investigation on 3D scaffold is summarized in table I. Alginate, collagen, and acellular tissue matrices (produced by decellularizing different tissues or organs) have special characteristics, including the capacity to produce hydrogels with 98-99% aqueous media at physiologic conditions.Testicular cells can develop and migrate more easily on hydrogel testicular matrices.The essential elements of testicular ECM should still be present in these matrices, while cells, bioactive cellular proteins, DNA, and substances that may interfere with the efficiency of culture should be eliminated

A
spontaneous circulatory connection between the host and the grafted tissue was developed as arteries sprouted from the transplanted component and joined the host's vascular system(49).Since every cell should be placed within at least 200 μm distance of a capillary, any tissue that has a longer distance must have a developed and sustained vascularization to survive, as results in extremely little quantities of testicular tissue (± 1 mm 3 ) experiencing a period of no blood supply, which is defined by hypoxia, even followed by a condition of reoxygenation, this tissue characterized by damage, as was previously shown for ovarian tissue transplantation(78).In culture-based research, platelet-derived growth factors, FGF, and vascular endothelial growth factors have been used in various studies to increase vascularization.Because of their short halflives, the dangers of distant dispersion, such as enhanced vascular permeability with the risk of International Journal of Reproductive BioMedicine Fath-Bayati et al. the encouragement of tumor neo-angiogenesis, hypotension, and vascular growth factors present substantial hurdles when employed in vivo.By employing encapsulating matrices and customized nanoparticles, these problems might be solved by the controlled release of physiologically active chemicals contained in the place of interest (79).
conditions are divided into 2 categories before and after puberty.There is currently no option to preserve the testis tissue except by using various techniques and in vivo or in vitro inducing differentiation in pre-puberty situations when a person does not have mature sperm.In humans, when prepubescent boys face cancer treatment, freezing immature testicular tissue containing SSCs can be suggested.In adulthood, fertility can be restored by autologous tissue transplantation or its SSCs, or in vitro maturation.So far, animal studies have been conducted with good results, but extensive and acceptable clinical trials have not yet been performed.Transplantation of testicular pure cells is impossible due to the risk of reintroducing cancer cells.
discussed, we tried to recognize the advantages and disadvantages of various plans and, utilize the best technique for the intended individuals in different conditions.Of course, researchers are still working on determining the best and most efficient strategy, and one of the main issues in this area is how to preserve and differentiate germ cells.

Table I .
Some studies on the use of tissue engineering in restoring spermatogenesis