medium serum free Archives - Gen9 Genetics
Cell therapies using stem cells are being explored in veterinary clinical practice. Among the different stem cells, mesenchymal / stromal stem cells (MSCs) are a cell type preferred by physicians and academics, in part due to their ease of isolation [1, 2]. MSCs are self-renewing post-embryonic cells that are capable of giving rise to a variety of parenchymal cells in vitro when stimulated with inducers [3]. MSCs are also clonogenic and form stromal progeny in vitro, express general fibroblastic markers, and, when transplanted, modulate the host immune system [4]. MSCs are transient cells that are believed to have a limited life span at the receptor [5]. Other potentially beneficial properties include the ability to host sites of damage and inflammation and secrete trophic factors that influence the repair of damaged tissues. However, the actual in vivo effects of manufactured human or veterinary MSCs are variable [3, 6, 7].
Although MSCs can be isolated from all postnatal tissues, usually fat tissue or bone marrow are major sources of MSCs due to their relative ease of isolation. Because their numbers in adult tissues are low, MSCs are usually expanded in culture to reach a sufficient number [8]. There are a variety of methods and media for the in vitro culture of MSC. Variations in isolation methods or culture conditions, such as culture reagents, culture vessels, and culture environment, contribute significantly to the heterogeneity of MSCs. Some means for both isolation and expansion of human or veterinary MSCs from fat tissue and bone marrow are described in the literature. They typically range from Minimal Essential Medium (MEM) to Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10-20% (v / v) Fetal Bovine Serum (FBS). FBS provides attachment factors, growth factors, and a host of other nutrients. The concentrations of these factors and nutrients in FBS vary widely between suppliers and can also vary between batches even when sourced from the same supplier. Therefore, the use of FBS containing uncharacterized components contributes to the heterogeneity of the number and quality of MSCs when switching between batches [9,10]. While this may not be an issue from an academic standpoint, for regulatory purposes, consistency in the quality of MSC batches is critical in the manufacturing process. Some of the growth factors present in FBS also promote stem cell differentiation [11]. FBS can also be a source of adventitious pathogens and contains serum proteins that have the potential to elicit an immune response in receptors. Safety, efficacy, consistency and reproducibility concerns make the proposal for a medium without FBS attractive.
To overcome some of the deficiencies associated with the inclusion of FBS in MSC culture, the use of autologous or allogeneic serum, plasma or platelet lysates is proposed for the culture of human MSCs [12]. Similarly, there are reports on the use of blood products for culturing canine MSCs [13]. However, autologous or allogeneic serum or blood products may not be practical for the expansion of canine MSCs because: large amounts of autologous serum may be required for the generation of clinically relevant amounts of MSC; Autologous or allogeneic serum derived from adult donors may not contain sufficient growth factors to support the growth of MSCs; and allogeneic serum is a potential source of infectious agents [13]. However, these FBS alternatives have the same potential to induce variability in cell culture as FBS.
While the concept of a predominantly serum-free medium devoid of animal components to eliminate the variability associated with FBS in MSC production is not new to the culture of human and rodent MSCs, the growth efficiency of MSC varies depending on the formulation of the MSC. medium [11, 14]. Likewise, the use of serum-free media developed for the isolation and expansion of human or rodent MSCs for the expansion of canine MSCs is often obtained with mixed results [14,15]. Therefore, the inconsistencies in the growth promoting potential of serum-free media developed for human MSCs in canine MSCs further suggest that single or growth-promoting nutrients are needed for the cultivation of canine MSCs.
Here we report the development of a serum-free medium for the in vitro expansion of canine adipose tissue MSC (Ad-MSC). We find that our medium s
Cell therapies utilizing stem cells are being explored in veterinary clinical practice. Amongst different stem cells, mesenchymal stem/stromal cells (MSCs) are a favored cell type by clinicians and academics alike partly because of their ease of isolation [1, 2]. MSCs are post-embryonic, self-renewing cells, which are capable of giving rise to a variety of parenchymal cells in vitro when stimulated with inducers [3]. MSCs are also clonogenic and form stromal progeny in vitro, express general fibroblastic markers and when transplanted, modulate the host immune system [4]. MSCs are transient cells that are believed to have a limited life span in the recipient [5]. Additional potentially beneficial properties include the ability to home to sites of damage and inflammation, and secrete trophic factors that influence the repair of damaged tissues. However, real in vivo effects of manufactured human or veterinary MSCs are variable [3, 6, 7].
Although MSCs can be isolated from every postnatal tissue, typically fat tissue or bone marrow are prime sources for MSCs due to their relative ease of isolation. Because their numbers in adult tissues are low, MSCs are typically culture expanded to attain a sufficient quantity [8]. A variety of methods and media exist for in vitro cultivation of MSCs. Variations in isolation methods or culture conditions such as culture reagents, culture vessels and culture environment contribute significantly to the heterogeneity of MSCs. A few media are described in the literature for both the isolation and expansion of human or veterinary MSCs from fat tissue and bone marrow. Typically, they range from Minimum Essential Medium (MEM) to Dulbecco’s Modified Eagle Medium (DMEM), which are supplemented with fetal bovine serum (FBS) at 10–20% (v/v). FBS provides attachment factors, growth factors and a host of other nutrients. Concentrations of these factors and nutrients in FBS vary greatly amongst suppliers and can additionally vary amongst batches even when obtained from the same supplier. Thus, utilizing FBS containing uncharacterized components contributes to the heterogeneity of MSC number and quality when switching between lots [9, 10]. While this may not be an issue from an academic stand point, for regulatory purposes consistency in the quality of batches of MSCs is critical in the manufacturing process. Some of the growth factors present in FBS also promote differentiation of stem cells [11]. FBS can also be a source of adventitious pathogens and contains serum proteins that have the potential to elicit immune response in recipients. Safety, efficacy, consistency and reproducibility concerns make the proposition of a medium void of FBS attractive.
To overcome the some of the deficiencies associated with the inclusion of FBS in cultivation of MSCs, use of autologous or allogeneic serum, plasma or platelet lysates are proposed for cultivating human MSCs [12]. Similarly, there are reports on the use of blood products for the cultivation of canine MSCs [13]. However, autologous or allogeneic serum or blood products may not be practical for canine MSC expansion because: large amounts of autologous serum may be required for generation of clinically relevant numbers of MSCs; autologous or allogeneic serum derived from adult donors may not contain sufficient growth factors to support growth of MSCs; and allogeneic serum is a potential source of infectious agents [13]. However, these FBS alternatives have the same potential for inducing variability in cell culture as FBS.
While the concept of serum-free medium predominantly devoid of animal components to eliminate variability associated with FBS in MSC production is not novel for cultivation of human and rodent MSCs, efficiency of MSC growth varies depending on the media formulation [11, 14]. Likewise, utilization of serum-free media developed for isolation and expansion of human or rodent MSCs for the expansion of canine MSCs is often met with mixed results [14, 15]. Thus, inconsistencies in growth promoting potential of serum-free media developed for human MSCs on canine MSCs further suggest that unique nutrients or growth stimulants are needed for the cultivation of canine MSCs.
Here we report the development of a serum-free medium for in vitro expansion of MSCs from canine adipose tissue (Ad-MSCs). We find that our serum-free medium efficiently supported both derivation and in vitro expansion of canine Ad-MSCs. Additionally, canine Ad-MSCs cultivated in this medium exhibited faster growth rates as measured by a lower population doubling (PD) time and a decreased lag phase as compared to canine Ad-MSCs cultivated in serum-containing medium.
Materials and methods
Media
Serum-containing medium.
Serum-containing medium consisted of DMEM/low glucose (Sigma, St. Louis, MO) dissolved into tissue culture grade water (Lonza, Walkersville, MD), filter sterilized and supplemented with 4 mM Glutamax (Life technologies, Carlsbad, CA), 1 x Antibiotic-Antimycotic (Invitrogen, Carlsbad, CA) and 10% heat inactivated fetal bovine serum from HyClone (Logan, UT) here after DMEM/FBS-H or Sigma (St. Louis, MO) here after DMEM/FBS-S or Dog serum (Equibiotech, Kerrville, TX) here after DMEM/DOG-S.
Commercial serum-free medium.
A commercial serum-free medium developed for expansion of human MSCs was purchased from RoosterBio (Frederick, MD). The composition of this media is proprietary and was not available to the authors.
Formulation of serum-free medium in our laboratory.
Typically, cells need nutrients (organic nutrients such as carbohydrates, lipids, amino acids, and vitamins; inorganic nutrients such as salts and trace minerals) and non-nutrient factors (such as hormones and growth factors), which are supplied by FBS, and a conducive environment (oxygen, humidity and temperature). Bearing in mind these requirements, we formulated a synthetic medium with defined components. To satisfy the nutrient requirements, we utilized basal media, which included DMEM (contains limited ingredients at higher concentration) and Ham’s nutrient mixture F-12 (contains a variety of ingredients at lower concentration) [16–18]. An equal powder mix (w/w) of these basal media would yield a medium balancing both concentrations and ranges of ingredients. In fact, DMEM/Ham’s nutrient mixture F-12 is often used as a starting formulation for proprietary media [19].
Powder media such as DMEM or Ham’s F-12 are typically supplemented with FBS to provide nutrients and growth factors that are either lacking or present at limiting amounts. A major component of FBS is albumin, which is usually present at concentrations varying from 20 to 50 mg/mL. Concentrations of other components of FBS, such as growth factors, amino acids, carbohydrates, lipids, and hormones are more variable. In addition, FBS also has many undefined components whose role in cell growth and metabolism is unknown. Because our aim is to replace FBS, we supplemented the powder media mix with the most critical components present in serum. FBS can contain enzymes such as lactate dehydrogenase (LDH), alkaline phosphatase (ALP); prostaglandins, endotoxin, hemoglobin, bilirubin, urea, creatinine, prolactin and other unidentified components. However, these components may not be important for cell culture therefore we did not add them to our serum-free medium. Table 1 details all the components in our serum-free medium.