Animal models
Research in biotechnology and immunology requires the use of various animal models. The fact that something can be done in the test tube (in vitro) with a cell line above, does not mean that it can be done in vivo in a living animal. For example, if large amounts of antiserum are needed, a rabbit, goat, sheep or horse might be the best animal. If one is working on a vaccine, the animal chosen must be susceptible to the infectious agent you are studying. Many animals, for example, are not susceptible to HIV infection. Such animals would thus not be good research models to study the efficacy of an HIV vaccine. Mice are the most common type of research animal used. They are easy to handle and have a rapid breeding cycle. Their immune system has also been extensively characterized. For some protocols on the use of mice click here.
The FDA Modernization Act 2.0 challenges the entrenchment in vivo animal modles in prelcinical work and suppots development of alternative models. And yet anotehr piece of legislation, the Biosecure Act, if assed, will significantly ipact the sources US researchers use to obtain theri animal models.
Types of Mice Models
Inbred Mouse:
Immunologists often work with genetically identical animals produced by inbreeding. This is advantageous in that one can control variation caused by differences in the genetic backgrounds of the mice. Repeated inbreeding for 20 generations usually yields an inbred strain whose progeny are homozygous at more than 98% of all loci. Hundreds of different inbred strains of mice are available. These strains are identified by a series of letters and/or numbers. Most of these strains are purchased by suppliers such as Jackson Laboratory in Bar Harbor, Maine.
Some common inbred mouse strains include : (1) balb/c (very docile) (2) AJmice (more aggressive) (3) black (aggressive) and (4) DBA/2 (very aggressive). Mouse genetics unit
Adoptive-Transfer Systems: In adoptive-transfer systems, the immune cells of the recipient mouse is inactivated by exposing the host to x-4ays which can kill almost all of its lymphocytes. If the host’s hematopoietic cells might influence the adoptive-transfer experiment, then even higher x-ray levels are used to eliminate the entire hematopoietic system. Thereafter the lymphocytes form the spleen of a donor can be studied without interference from the host lymphocytes.
Scid Mouse: “Scid” stands for severe combined immunodeficiency disease. Scid mice fail to develop mature T and B cells and are thus severely compromised immunologically. The absence of these cells enables SCID mice to accept foreign cells and grafts from other strains of mice and other species. An autosomal recessive mutation resulting in SCID developed spontaneously in a strain of mice called CB-17.
Scid mice have been very useful in the study of human lymphocytes. For example, one can transplant human thymus and lymph node tissue under the kidney capsule of the SCID mouse and inject the mouse with human fetal liver cells (stem cells). Those stem cells will then migrate to the human thymus where they will mature into T cells.
Transgenic Mice: are mice which have a gene insertion, deletion or replacement. The foreign or modified genes that are added are called “transgenes.” There are two main methods to create transgenic mice. The first relies on random insertion of the gene into the mouse. The second rlies upon homologous recombination.
Appropriate Doses to Use in Mice Studies
The precise does to be employed in an antibody formualtion will depend on the route of administration and the seriousness of the disease or disorder and hsould be decided according to the jugment of the practitioner and each patient’s circustances. Effective doses may be extrpolated from dose reponse curves derived from in vitro or animal model test systems. Relveant cirumstances to be considered include the choice of composition to be adminsitered, the age, weight and response of the patient and the severity of the pateint’s symptoms. For example, the actual patient body weight may be used to calculate the dose of the formulations in milliliters (mL) to be admistered as caculated by the formula: Dose (mL)= [patient weight (kg). times dose level (mg/kg)/drug concentration (mg/mL) (Spuler, US2009/0041764; see also Bell (US 2009/0220508))
Body surface area (BSA) normalization method: Animal does should not be extrapolated to a human equivalent does (HED) by a simple conversion based on body weight. For the more appropriate conversion of drug doses from animal studies to human studies, the body surface area (BSA) normalization method should be used. The formula is the following:
HED (mg/kg) = Animal dose (mg/kg) multiplied by Animal Km/Human Km
The Km factor for an adult human is 37 and for a child is 25.
The Km for a mouse is 3
For reference and values for other experimental animals see Reagan-Shaw (“Dose translation from animal to human studies revisited” FASEB J. 22, 659-661 (2007).
See also “Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers” US Dept Health and Human Services Food and Drug Administration (July 2005).
Example: To calculate an equivalent human dose of 40 mg/kg to a mouse, one would multiple this # by the ratio of of the Km for the mouse (3) divided by the Km for an adult (37) (40 x (.2627) = 3.24 mg/kg equivalent human dose which equate to a 243 mg dose for a 75 kg person. It would not be proper to simply take the 3.24 mg/kg and multiple this by 75 kg to get a doese of 3,000 mg.
Alternatives to Mice Models:
Animal models are imperfect models. Alternative models includes organoid systems, which incorproate human cells and tissues. But tissues thare harvested from people are limited in quantity. Also, these systems may lack some of the cell types and the vascular features found in biopsied tissue.
3D printing:
Carcinotech is using 3D printing tumors that more closely mimic human biopsies. They have many different cell types in one printed tumor and a whole immune panel can be profiled. The 3D printed tumors also offer consistency as well as capital conservation. During the early stages of drug discovery, a company can spend 7-8 million on preclinical testing per compound. They can reduce this figure by more than one half because the models provide much more data. Scientists could use 50-200 mice for efficacy studies or they could replace them with 100 pateint profiels using 3D printed tumors. The use of printed tumors, organ-on-a-chip-devices and other modesl may get a boost from teh FDA Modernization Act 2.0, which was signed into law in 2022. It authorizes the sue of certain latenratives to animal models in drug testing.
Organoids:
Organoids are dervied from pluripotent stem cells or isolated organ progenitors that differentiate to form an organ-like tissue exhibiting multiple cell types that self-organize to form a structure not unlike the organ in vivo. Because organoids can be grown from humn stem cells and from pateint derived induced pluripotent stem cells, they ahve the potential to model human development and disease. In addiiton, they ahve potential for drug testing. Orgaonids have beome increasingly popular as tissue odels since they can retain the gentic and phenotypic heterogeneity of the orginal tissue.
Organoids can be assembled or allowed to self-assemble. In either case, they are typically cultured with specific growth factors and extracellular matrix proteins. This approach was pioneered in 2009, when researchers generated the first 3 D organoid culture form adult stemp cells.
–Companies/products:
—Corning Matrigel Matrix: is a solubilized basement membrane preparation. Rich in extracellular matrix proteins such as laminin and collagen, Matrigel Matrix is sued to produce hydogels that support organ/tissue architectures.