Umbilical Cord Blood and Cord Tissue Bank as a Source for Allogeneic Use

Recently, umbilical cord blood (CB) has received attention as the allogeneic optimum source for immunotherapies. More recently, the umbilical cord (UC) has been rapidly utilized as an abundant source of mesenchymal stromal cells (MSCs), which migrate toward the inflammatory and damaged tissue to subside the inflammation and support tissue repair. Both CB and UC can be provided “off-the-shelf” cell products for immunotherapies and regenerative medicine. As biomedical wastes, CB and UC can be obtained noninvasively without any risks to the donor. CB cells and UC-derived MSCs (UC-MSCs) also have higher proliferation potentials than other cells obtained from adult tissues. In addition, UC-MSCs are less immunogenic and have significant immunosuppressive ability. Several clinical trials with CB or UC-MSCs have been conducted based on these advantages. The establishment of a stable supply system of CB and UC-MSCs is critical now for their utilization in regenerative and immune cell therapies. We have thus established the cord blood/cord bank, “IMSUT CORD,” as a new type of biobank, to supply both frozen CB and UC tissues and derived cells for research and clinical uses. In this chapter, we will introduce the overall flow from collection to shipment and discuss several issues that need to be resolved in unrelated allogeneic stable supply system.


Introduction
Umbilical cord blood (CB) has been utilized as a source of hematopoietic stem cells for three decades. It could potentially also serve as the optimum source of immune cells, such as mononuclear cells (MNC), regulatory T cells, NK cells, and mesenchymal stromal cells (MSCs) with or without genetic modifications, for immunotherapy and neurogenic regeneration in some cases. In addition, UB could be prepared as readily available products [1].
It is well-known that human mesenchymal stromal cells (MSCs) can be harvested from various tissues that include the bone marrow (BM) [2], cord blood (CB) [3], adipose tissue [4], placenta [5], and umbilical cord (UC) [6]. Recently, clinical trials using MSCs for various diseases have been conducted, and some of them were approved. The BM is considered the traditional source of MSCs, and the characteristics and applications of BM-derived MSCs (BM-MSCs) have

Cord blood and umbilical cord collection
There are many public and private CB banks in the world, in which procedures are nearly standardized intended for hematopoietic stem cell transplantation (HSCT) as shown in Section 5. The procedures include informed consent acquisition from the mother, collection of CB, processing, storage, to shipment, which have been already established. In the case of UC bank for unrelated allogeneic uses, the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) which issued ICH Q5A as the regulation of materials for biological products requires the second blood test from the baby's mother, to deny viral infection in window period at delivery. Figure 1 shows the overall process of banking in the mother's side. We deal with both CB and UC. In

Cord blood processing
Among the processing methods to obtain nucleated cells from CB for hematopoietic stem cell transplantation, the hydroxyethyl starch (HES) centrifugation method (HES method) is the most efficient and common. The HES method originated from the New York Blood Center CB bank [8]. Recently, automated CB processing SEPAX® (GE Healthcare Life Sciences) [9] and AutoXpress Platform® (Cesca Therapeutics, Inc.) [10] have been developed. For CB cryopreservation, DMSO and Dextran 40 together with CB-plasma are used worldwide [8].
On the other hand, no processing method of fresh or frozen CB not for hematopoietic stem cell transplantation has been settled as standard. The use of mononuclear cells (MNCs) obtained by the Ficoll-Paque centrifugation method (Ficoll method) or cell sorting with antibody-conjugated magnetic beads might be a new candidate for further processing and culture method. CB processed by HES method resulted in whole nucleated cells including neutrophils, monocytes, lymphocytes, and nucleated red blood cells with some amount of red blood cells (RBC). The recovery rate of hematopoietic stem cells and mononuclear cells processed by HES method is superior to those by Ficoll method. That is why HES method is introduced by CB banks in the world [8]. However, neutrophils in the nucleated cells and residual RBC may cause the aggregation resulting in the difficulty of further processing, when the frozen and thawed cells are diluted with large volume of isotonic solutions such as medium. Only frozen-thawed CB nucleated cells can be diluted with dextran and albumin/saline solution [8]. On the other hand, frozen-thawed MNCs processed by Ficoll method does not require such a special solution and can be diluted with medium and PBS, although the recovery rate of MNCs from the fresh CB by Ficoll method is less than that by HES methods.

Umbilical cord processing
There are diverse procedures and culture methods for the isolation of MSCs from the various compartments of UC, such as Wharton's jelly, veins, arteries, UC lining membrane, subamnion, and perivascular regions [12]. The isolation methods of MSCs from the Wharton's jelly, vein, and arteries of UC are reported previously, although the marked differences were not found as far as the 10% fetal bovine serum (FBS) and α minimum essential medium (MEM) [13]. There are several papers to obtain MSCs from whole UC versus Wharton's jelly [14] or different parts of the same UC [15], but we suggest that to process from whole UC seems sufficient and simple for further processing [15]. Despite the wide range of isolation and culture procedures, the different groups seem to agree on the cryopreservation of UC tissue [16] and explant method [17] followed by the harvest of migrating cells from tissue. However, large-scale culture methods remain to be determined. Figure 2 shows the example of scheme of CB and UC collection and process and shipping to clinical use.

Cryopreservation of UC tissue
It is known that the UC tissue can be frozen in a cryoprotectant. This possibility of cryopreservation is the advantages of UC tissue for both clinical and research uses. The reasons of the advantages are: 1. UC tissue processing can be started after the donor's health and infection statuses are confirmed well. This leads to initial cost-effectiveness because unnecessary works using inappropriate materials are eliminated. In addition, we can thaw a small amount of the UC to survey, before culturing MSCs in a large scale.
2. Storage of the tissues of origin allows us to keep traceability and to check the quality as the biological resources at a later date.
3. When new reagents or techniques were developed in the future, we can isolate novel cells from the cryopreserved UC tissues.
4. If the donor, the baby, has diseases that can be treated with autologous cells, including iPS cells or gene-modified cells, or autologous UC-MSCs, the cryopreserved UC tissues would be the appropriate source.
Several animal serum-free cryoprotectants containing 5-10% dimethyl sulfoxide (DMSO) are available. Whether the use of serum originating from animals, such as fetal bovine serum (FBS), is required, is critical, because it adds the risk of the transmission of zoonotic infections, immunological reactions, and additional regulatory issues [18]. There are several reports of cryopreservation of the UC tissue, using serum-free and xenogeneic animal-free (xeno-free) cryoprotectants. Ennis  UC tissue in 10% DMSO and 0.2 M sucrose solution, but the cumulative cell yield derived from the frozen-thawed UC-MSCs in their solution was inferior to that of fresh UC-MSCs [20]. We previously reported the cryopreservation of UC tissue, with no impact on viability, using a serum-and animal origin-free cryoprotectant, STEM-CELLBANKER ® [16]. We demonstrated that cells derived from UC cryopreserved in this manner retained the phenotypic characteristics of MSCs, including the immunosuppressive activity in allogeneic mixed lymphocyte reactions, as well as differentiation potential. As shown in Figure 2, with the cryopreservation of UC tissue, UC processing might be altered.

Improved explant method
There are two major approaches after frozen-thawed UC tissue: explant and enzymatic digestion methods. Frozen-thawed UC tissue is manually minced into small fragments approximately 1-2 mm 3 in size. Mincing is preferred to using a surgical scalpel or the use of an autologous mixer. These fragments are aligned and seeded regularly on a tissue culture-treated dish. After the tissue fragments attach to the bottom of the dish, culture media is added, slowly and gently in order not to detach the fragments [21][22][23][24]. Media is then refreshed every 3-7 days for 2-4 weeks until the fibroblast-like adherent cells reach 80-90% confluence. Subsequently, adherent cells and tissue fragments are rinsed once with PBS, detached using trypsin, and washed with media. The culture is then filtered to remove tissue fragments. The disadvantage in the explant method is that tissue fragments often float in media, resulting in the poor recovery of cells. To protect the exfoliation of tissue fragments from the bottom of the culture dish, we introduced stainless steel mesh (Cellamigo ® ; Tsubakimoto Chain Co.) shown in Figure 2, No. 8. In this manner, we can plate source tissue more quickly and harvest more MSCs. In addition, the incubation time required to reach 80-90% confluence is reduced [17].
In the enzymatic digestion method, UC is minced into small pieces and immersed in the media containing enzymes such as collagenase, or a combination of collagenase and hyaluronidase with or without trypsin [21,[24][25][26]. The cells dissociated by the enzymes are then cultured until they reach full confluence. However, the digestion method is costly and time-consuming and may result in decreased cell viability due to lytic activity and varying sensitivity of the cells to collagenase. In addition, the initial harvested cells include more of the other types of cells compared with that harvested using the explant method.

Large-scale expansion and harvesting the cells
It is critical to consider how much we can expand the UC-MSCs to allow allogeneic "off-the-shelf " industrial availability, because the proliferation of adherent cells needs a large surface area. The conventional method uses multilayered flasks, and the cells are cultured in incubators installed in cleanrooms. These multilayer flasks can consistently support the expansion of UC-MSCs, and the state of cell confluence can be examined under the microscope. However, this method requires the considerable involvement of operators because the processes of seeding, refreshment, passage, and harvest require individual and manual works. Several companies have introduced the spinner bioreactor with a microcarrier made of plastic, dextran, denatured collagen-coated beads, and other components. The bioreactor system may reduce the number of operators required and may allow to reduce the clean levels of facility since it is a closed system. On the contrary, several critical problems of the bioreactor system exist. The cost of equipment is high and it is difficult to evaluate cell proliferation environment such as pH, lactate, and so on. When some microcarriers are torn off by spinner, or undigested microcarriers are residual in the final products, we have no ideas to remove the residual microcarriers completely. Recently, a plastic bag bioreactor system with a microcarrier in gentle locking was reported [27]. The most critical problem is that the cells produced by the flask-based culture method may be different from those by bioreactors. Harvesting cells on a large scale is still not easy. Recently, filter-based cell concentration and washing systems were introduced (https://www.kaneka.co.jp/en/business/healthcare/ med_006.html, KANEKA, Japan). Automatic cell packaging may be also required in large-scale expansion.
The academic culture level such as IMSUT CORD is at small to middle scale. Only the company may have the ability to expand the cells at extra-large scale and maintain to control and supply the cell product for clinic constantly.

Long-term cryopreservation
Master and product cells of UC-MSCs for clinical use are usually required for long-term cryopreservation, together with records on the donor infant and the mother. There are several cryoprotectants for long-term cryopreservation. The most popular cryoprotectant consists of 5-10% dimethyl sulfoxide with human albumin. Recently, serum-free cryoprotectants, described in Section 4.1, have been commercialized and are thought to be more ideal compared to those containing humanderived serum. In addition to cryoprotectants, it is important to build an adequate record preservation system. Those who manage the long-term cryopreservation should preserve the records that include the documentation relating to the collection including donors, processing, results of quality tests, and instruments directly related to the products. The kinds of records and the length to be preserved are in accordance with the bank policies and standards and the corresponding domestic laws and regulations. It is necessary to discuss how long we should or we can cryopreserve CB and UC tissue, UC master cells, and product cells, in the technical and ethical aspects. In the technical aspect, the cell-preserving vessel to accommodate the cell suspension for long-term freezing should be durable in a liquid nitrogen. In the ethical aspect, we do not expect whether the babies can recapture their ownership of CB and UC even though their mother waived the ownership of them, when they grow up to be adult.

Quality and safety assurance of UC-MSCs
The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposed the minimal criteria for defining human MSCs [28,29]. First, MSCs must be plastic-adherent when maintained in standard culture conditions. Second, MSC must express CD105, CD73, and CD90 but not CD45, CD34, CD14 or CD11b, CD79α or CD19, and HLA-DR surface molecules. Third, MSCs must differentiate into adipocytes, chondroblasts, and osteoblasts in vitro [30][31][32]. Immunosuppressive effects have now become the most popular property of MSCs for potential clinical use [12]. Defect in HLA-class II expression with negative CD80 and CD86in UC-MSCs even in the presence of inflammatory cytokines such as IFN-γ can theoretically rescue them from immune recognition by CD4+ T cells [33]. MSCs can also inhibit proliferation of and cytokine secretion by immune cells, as well as alter subtypes of macrophage from M1 to M2 in vitro [34][35][36][37]. This immunomodulation is linked mainly to soluble factors such as indoleamine 2,3-dioxygenase (IDO), PGE2, and HLA-G5 [38], hepatocyte growth factor, and transforming growth factor-β1 released from MSCs [39]. Further quality tests are dependent on clinical applications and characteristics of MSCs.
The safety tests required differ according to the risks of clinical applications. For example, the tests of CB banking for hematopoietic stem cell transplantation are different from those of UC-MSCs. Donor-recipient relation of the former is one-to-one, and the risk is limited to one patient. On the other hand, that relationship of the latter, as UC-MSCs master cells and product cells, is one-to-many, so hundreds of patients may suffer health injuries by one donor. Thus, the vials of UC-MSCs are tested thoroughly at a designated time not only known viruses but also unknown viruses. Those tests should follow the local, national or international applicable laws and regulations. More precise safety tests for CB and UC shall be described elsewhere for the respective products for clinical application.

Standards and guidance for CB and UC from collection to release
There are international standards/guidance for CB collection, banking, and release of hematopoietic stem cell transplantation, such as the Foundation for the Accreditation of Cellular Therapy (FACT)/NETCORD [40], American association of Blood Banks (AABB), US Food and Drug Administration (FDA) shown in Table 1, and local standards or regulations under the applicable laws in respective countries. A CB/UC bank, facility, or individual should implement if the standard of practice in the community or applicable law establish additional requirements. International standards/guidance for biobanking process for UC collection, processing, culture, and release has not been settled, but collection and banking protocols can follow the CB banking standards and good tissue practice. Each CB/UC bank, facility, and individual should analyze its practices and procedures to determine whether additional standards apply. Compliance with the standards is not an exclusive means of complying with the standard of care in the industry or community or with local, national, or international laws or regulations [40]. Allogeneic public CB banks requested US FDA accreditation with FACT/NETCORD or AABB in the USA, while the CB banks in Europe (EU) required FACT/NetCord  with additional requirements like FACT/JACIE standards, when it is requested by the respective national regulation affairs. There are many private or private-public combined CB banks in the world, which tend to follow the AABB standards and have the inspection and accreditation (http://www.aabb.org/sa/facilities/celltherapy/Documents/AABB-Accredited-Cord-Blood-Facilities.pdf).

Special issues of CB and UC bank system for allogeneic use
The number of clinical trials using CB and UC-MSCs in the fields of immune cell therapies and regenerative medicine has been increasing. On the other hand, CB as a source of hematopoietic stem cell transplantation is less used recently, because the cell number is limited, the engraftment of HSC is delayed, and HLA haplo-identical HSCT is induced and controlled. These clinical trials are aimed uses that include severe acute graft-versus-host disease (GVHD) treatment, rapid engraftment of HSCT, and the prevention of severe acute GVHD. Clinical trials using CB-and UC-MSCs are summarized in Tables 2 and 3, respectively. We started a sponsorinvestigator clinical trial using UC-derived MSCs for patients with treatmentresistant severe acute GVHD supported by the research fund of the Japan Agency for Medical Research and Development (AMED). Consistent supply is the critical key to conduct clinical trials and for marketing. For the stable supply of frozen CB and UC, or UC-derived MSCs, we have established a CB and UC bank, named IMSUT CORD, in our institute. This bank also provides CB and UC-MSCs for immunotherapy and regenerative medicine products to hospitals and pharmaceutical companies shown in Figure 2. The bank also provides frozen CB, frozen UC, master cells, and the cells after master cells as an intermediate products requiring further processing or more culture in the companies.
The following are also the major points for managing CB and UC banking. First, to build an adequate quality management system to serve the resource of cell therapy products, we have introduced the concept of the ISO 9001 and obtained its certification, and as a result, we introduced the concept of PDCA cycle. Second, involvements of various kinds of specialists must be considered. There are many procedures, such as collection, obtaining informed consent, application to ethics review committee, and document management. Third, health check and infection test of the donor's mother are required to ensure that no infection is detected after window period of infections. In this process, both traceability and personal information protection must be satisfied. Fourth, we respect the right of decision to donate, rejection, or withdrawal. Donor's mother should be explained the policy of the bank that the consented withdrawal time is set at the initiation of processing for clinical use. Although the CB and UC belong to the baby, we obtain informed consent from the donor's mother as guardianship and ownership are asked to be transferred to the bank. Fifth, there is also the issue of how long the UC tissue and UC-MSCs can be cryopreserved. For example, in the Japanese public CB bank for HSCT, the CB is cryopreserved for 10 years as a clinical grade of HSC source. After this period, they are used for basic research or preclinical studies or discarded if they are not used for research. A cryopreservation period of 10 years for UC and UC-MSCs may be the first threshold to be checked. In addition, we disclose the information in website for the mothers who have not been explained about the new researches or new clinical trials at the first IC acquisition. Lastly, because unlike CB, the UC is a tissue considered as a part of the perinatal appendage, we must follow the tissue transport and medical disposal/waste regulations under the applicable laws or local rules and ethical standards.