The Use of Human Tissues for Research: What Investigators Need to Know

Introduction

The purpose of this article is to help researchers transition from the use of animals to the use of human tissues in their research. The use of human tissues can help ensure that any preliminary findings obtained in animals are relevant to human systems. As we discuss in this paper, the use of human tissues in research is critical for the validation of findings obtained in animals. The use of human tissues has led to major scientific and medical advances. However, investigators wishing to transition to the use of human tissues may not know how to identify appropriate high-quality sources of biospecimens, or understand the requirements related to their use. We discuss these issues, with the hope that a better understanding of these issues will assist researchers in their transition from the use of animals to human tissues. This should lead to quality findings that will advance our understanding of biology and science, and contribute to major developments in medicine and technology.

Advantages of using human tissues in research

The goal of most biomedical research is to increase our understanding of the mechanisms underlying a particular disease, with a view to improving the available cures, therapies and management strategies. Frequently, cell lines and animal models are used to develop preliminary data to achieve these goals. However, the use of cell lines and animal models has disadvantages, and the use of human tissues may help achieve these goals more rapidly. For example, cell lines develop and accumulate many molecular changes, such as chromosomal instability, compared to the original human tissues from which they were developed, so that they may not then accurately reflect the original tissue. ¹ Cell lines can also be misidentified and contaminated by other cell lines. ² In addition, the genomic responses, immunology, and other biological responses of animals, may vary significantly from those of humans. ³ Because of this, results from animal models and cell lines will usually have to be confirmed in human tissues.

Advances in science and medicine made possible through the use of human tissues

Almost all advances in medical care are based on research in which the use of human tissues has played a significant role. The recent increased availability of human tissues for research has led to many seminal discoveries in science and major developments in medical care, as we describe in detail subsequently.

Information obtained from human tissues has been critical to the diagnostic characterisation and subtyping of most cancers. For example, most breast cancers are morphologically described (lobular, ductal and mixed), as well as molecularly subtyped. Similarly, the diagnosis of lymphomas has recently been changed, and is currently based on both morphologic characteristics and molecular features. ⁴ Indeed, morphologic subtyping and determination of molecular features have become crucial in the diagnosis and therapy of many types of cancers.

Human tissues have played an essential part in many important developments in precision medicine. Precision medicine is a type of medical care in which characteristics of patients are combined with features of their diseases, in order to develop specific and individual patient-focused therapies.^5,6 Although ‘precision medicine’ is a relatively new term, this approach to medical care has been used for several decades, specifically to treat breast cancer. Physicians have long been targeting the molecular characteristics of breast cancers for treatment — for example, by blocking the oestrogen receptor. This therapeutic approach was developed through the use of breast cancer tissues, and, currently, the molecular features of each individual patient’s tissue are evaluated, in order to determine whether anti-oestrogen receptor therapy is appropriate.

In another example, human tissues were used to identify the human epidermal growth factor receptor type 2 (HER2) as a molecular marker of poor prognosis in breast cancer. ⁷ Subsequently, HER2 became a target of precision medicine in both breast and gastric cancers. Human tissues were used to test the HER2 antibody, Herceptin^®, and its subsequently-developed biological equivalents, such as Kanjinti^® as well as anti-HER2-based drug conjugates.^8,9 Similarly, the identification of HER2 overexpression in gastric cancers, as well as the sensitivity of HER2-positive gastric cancers to anti-HER2 therapy, drove the development of additional precision medicine-based therapeutic approaches for gastric cancer. ¹⁰

A more recent, tissue-based advance in precision medicine is the development of immunotherapy. Studies on human tissues have demonstrated that some cancers, such as melanoma and lung cancer, develop in an environment in which the immune system is suppressed. Two categories of immunotherapy have been characterised by utilising human tissues — one type involves the general stimulation of the immune system, and the other blocks the action of immune checkpoint inhibitors. A specific example of the latter is a strategy focused on the programmed cell death protein 1 (PD-1), which is expressed on T-cells. When the checkpoint inhibitor, PD-1, is activated, PD-1 decreases T-lymphocyte activation, cytokine secretion, and proliferation and survival of the T-cells expressing PD-1. Some cancer cells express the ligand for PD-1 (PDL1). When PDL1 binds to PD-1, it inhibits the immune response directed toward the cancer cells. ¹¹ Multiple antibodies have been developed to inhibit the PDL1 and PD-1 interaction. These have proved to be effective against PDL1-positive melanomas, lung cancers and other cancers.^12,13 However, some tumours are not very responsive to this type of immunotherapy.

Sources of human biospecimens

Human tissues that are used in research may involve a variety of cell types and preparations, and may be collected in a number of different ways. In addition to solid tissues, human biospecimens that are often collected and used for research include whole blood and blood components such as serum, plasma and buffy coat. They may also include other bodily fluids, such as urine, saliva/sputum and joint fluids, as well as cellular preparations (e.g. fine needle aspirates). Other types of biospecimens often used in research include cellular derivatives, such as DNA and RNA, as well as immortalised cell lines, organoids and patient-derived xenografts.^14,15

Human biospecimens that are used for research may be collected in a variety of ways. Often, remnant (surplus) biospecimens, that were originally obtained during the course of routine care, can be used for research, as long as the research use does not affect any future care of the patient. Biospecimens can also be obtained specifically for research with consent from the participant through a specific intervention (such as a blood draw, oral swab or urine collection), or collected for research purposes during a clinical protocol or as part of a clinically necessary procedure. For example, additional biopsy samples for research purposes may be taken at the same time as a clinically indicated biopsy. Finally, biospecimens can be obtained for research from cadavers or autopsies, or from unused tissues that may have been initially collected for transplant purposes. In all cases, biospecimens for research must be obtained following the relevant ethical and regulatory requirements, which in most cases requires consent from the donor/research participant.^16–18

What is a bioresource and what types of bioresources exist?

Biospecimens and associated data are collected and stored by entities often referred to as ‘biobanks’ or ‘biorepositories’, which are also responsible for making the biomaterial available for research. As defined by the International Society for Biological and Environmental Repositories, ¹⁹ a biorepository or biobank is: “An entity that receives, stores, processes and/or distributes specimens, as needed. It encompasses the physical location as well as the full range of activities associated with its operation”. A more recent term used to describe a biobank is ‘bioresource’. ²⁰ We prefer to use this term because it emphasises the important role of serving as a resource for the research community rather than simply storing and banking biospecimens.

A variety of different bioresource models provide biospecimens for research. These have been discussed in more detail in previous publications.^20,21 The most common bioresources serving typical investigators utilise: the tissue procurement model; the classic biobanking model; the population-based model; or a combination of classic biobanking and tissue procurement models.^21,22 As discussed subsequently, each of these models might be better suited to different kinds of research.

How to find an appropriate, good quality bioresource

While numerous bioresources exist, researchers are often unaware of how to find them. A number of tools exist to help researchers find suitable bioresources for their work. This includes the NCI Specimen Resource Locator, ²⁸ the ISBER International Repository Locator ²⁹ and the BBMRI-Eric Negotiator ³⁰ — a communication platform for bioresources and researchers requesting biospecimens and/or data.

One important bioresource supporting basic and developmental researchers who may be wishing to transition from the use of animal models to research on human tissues is the Cooperative Human Tissue Network (CHTN). ²³ The CHTN was established in 1987 to provide increased access to human biospecimens to accelerate discoveries in cancer research. However, the Network supports a wide range of research on human tissue, including research on other diseases. CHTN staff work closely with individual researchers to tailor collection processes and procedures to meet their precise requirements. All biospecimens that are collected must meet strict quality assurance/control standards, to ensure that the biospecimens are of high quality and fit for the intended purpose.^22,23

In addition to bioresources established and maintained by academic medical centres, there are multiple national and international commercial companies that provide human tissues to support biomedical research. These companies usually collaborate with medical sites or organisations from which they obtain the remnant tissues that they provide. Sometimes these companies specialise in only certain categories of tissue. For example, some companies collect blood and blood products for medical use from paid individuals; based on a fee, these individuals will provide biofluid aliquots to support research. The non-profit Red Cross may also provide selected blood biospecimens for research. Anabios specialises in cardiovascular and neuropathological tissues. A number of companies (e.g. Bocabio, Anabios, iSpecimen) can supply, or act as a broker to help researchers obtain, various biospecimens — including biofluids and solid tissues, especially as formalin-fixed paraffin-embedded (FFPE) blocks or sometimes as frozen biospecimens. Various extents of clinical and demographic data can also be provided, depending on the company. The main disadvantage of this type of commercial tissue supply is the high cost and, sometimes, inadequate quality control. A range of commercial companies providing human tissues can be found on the web.

An important aspect of using human tissues in research is tissue quality. Bioresources use a number of approaches to assure high quality and fitness-for-purpose of the biospecimens they provide. This includes establishing a rigorous quality management system (QMS) with demanding quality control (QC). The QMS must utilise an SOP for each activity and perform frequent audits to ensure that the SOPs are being followed. One aspect of the QMS is QC, which validates the diagnostic and other characteristics of each actual biospecimen provided to an investigator. For example, in the case of cancer biospecimens, QC data such as the percentage of tumour nuclei in the biospecimen, as well as the levels of necrosis, fibrosis and mucin, should be provided.^22,31,32

In order to assure high-quality biospecimens and data, bioresources often follow best practices, such as those developed by the International Society for Biological and Environmental Repositories (https://www.isber.org/). ³³ The document, ISBER best practices: Recommendations for repositories fourth edition, provides evidence-based or consensus-based best practices for the collection, storage and distribution of biospecimens for research.^34,35 International standards have also been established, including ISO 20387:2018 General requirements for biobanking,^36,37 and these are beginning to be implemented by bioresources. ³⁸ In addition, some bioresources may be accredited by the College of American Pathologists Biorepository Accreditation Program. ³⁹ These best practices, standards and accreditation programmes help to ensure that the biospecimens and associated data obtained from bioresources are of high quality and fit for their intended purpose.

Sources of normal tissues

Normal tissues may be difficult to obtain. Bioresources can collect normal tissues from autopsies and donated cadavers, but tissues affected by comorbidities should be avoided. When tissues are removed surgically, some tissues may be considered ‘normal’; these are tissues not involved in the disease processes being treated by the surgery. In the case of cancer, the organ affected is not considered normal. For breast cancer, the tissues of neither breast can be considered to be normal but adjacent skeletal muscle can be classified as ‘normal’, provided that it has not been invaded by the breast cancer. Population-based studies may serve as good sources of normal tissues. These include the National Health and Nutrition Examination Survey (NHANES), which aims to evaluate the health and nutrition of adults and children in the USA over time. ²⁵ Not all biospecimens that are collected are typically used by NHANES, and thus may be available to external investigators. Similarly, normal tissues that are not informative to the trial are available in the bioresource associated with the Prostate, Lung, Colon and Ovary (PLCO) Cancer Screening Trial; these biospecimens are likely to be available to investigators at no cost other than shipping. ⁴⁰

Biospecimens from rare diseases

When investigators need tissues from individuals with a rare disease, they might have difficulty in obtaining them, as most bioresources are unlikely to have such biospecimens in their inventories. Advocates for some rare diseases have recognised this as a significant problem, and often have an associated advocacy organisation that will fund the establishment of a bioresource for diseased tissues. Advocacy organisations might also encourage their members to donate remnant tissues of the rare disease, as well as matching uninvolved tissues, to the bioresource. Examples include the Chordoma Foundation Biobank; ⁴¹ other advocacy organisation-funded bioresources can be found on the web.

Requirements for access to human biospecimens

Bioresources often have a variety of requirements that must be met, in order to access the biospecimens and data that they provide. Some bioresources may restrict who can have access to the biospecimens; in this respect, they can either be ‘open bioresources’ or ‘closed bioresources’.

An open bioresource provides biospecimens to any investigators who meet its standards and requirements. It can often serve the important role of providing a biospecimen to supplement a biospecimen request that another bioresource, open or closed, cannot completely fulfil. On the other hand, a closed bioresource typically provides biospecimens only to investigators at the institution which is financially responsible for the bioresource; however, other investigators may be designated to have access. Thus, a closed bioresource is aware of the requirements of its potential users and can tailor its operations, including the collection of needed biospecimens, based on their stated and perceived needs. Operations can be adjusted when there are new investigators, or when new projects are developed by existing investigators. A closed bioresource may meet most of the requests by its investigators, for biospecimens with characteristics that are not too restrictive. For complex requests for biospecimens that a closed bioresource cannot adequately fulfil, open bioresources, such as the Cooperative Human Tissue Network (CHTN), ²³ can often supplement local biospecimen needs.

Bioresources often have requirements for access to the biospecimens and data that they provide, to ensure that the biospecimens are used in ways that are ethically appropriate and scientifically sound. This can include a requirement for some sort of review and approval by an ethics review committee. In addition, an internal review by the bioresource, sometimes by a biospecimen utilisation or similar committee, is often undertaken to ensure that an investigator’s request is an appropriate use of the bioresource and that it is consistent with biospecimen availability. Some bioresources require the researcher receiving the biospecimens to collaborate with the bioresource investigators.

As a best practice strategy, researchers and their institutions may be asked to sign an investigator agreement that defines the responsibilities and obligations of the recipient researcher and the bioresource. These agreements, which might include a Material Transfer Agreement and/or Data Use Agreement, aim to document the obligations of the researcher to protect the privacy of the individuals from whom the biospecimens are obtained and the confidentiality of their data, to use the biospecimens only for the approved project, and to agree not to share the biospecimens and data with third parties without approval of the bioresource. ¹⁹ These agreements may include other terms and conditions for use of the biospecimens and associated data (e.g. intellectual property rights), and might request that the investigators acknowledge the bioresource in any publications arising from the use of the tissue. These requirements help to ensure that investigators are using the biospecimens appropriately and help document the advances made possible from biospecimens obtained from the bioresource. This is important for the bioresource, so that it can demonstrate its value to funding agencies and sponsors in order to justify continued funding and provide sustainability for the bioresource.

Requirements for access to biospecimens can vary considerably from one bioresource to the next. Information about the types of biospecimens available from a bioresource, the requirements for access, and the application process are usually found on the bioresource’s website.

Operating a bioresource is expensive, and few bioresources are self-sustainable. Therefore, many bioresources charge a fee for biospecimens to cover the cost of procuring, annotating and distributing them for research. ‘Selling’ biospecimens for profit is generally considered unethical, although charging a fee for cost recovery is considered acceptable. ⁴² The cost per biospecimen charged by a bioresource depends on the efficiency of its operations and the number of biospecimens distributed. For example, if the costs of operating a small bioresource are US$200,000 per year and only 100 biospecimens are distributed, the actual cost of collecting, annotating and storing a biospecimen might be in the region of US$2,000. This obviously is too expensive for an academic investigator to pay. The decision on charge back fees is typically decided based on what the bioresource decides academic investigators can afford to pay; this is possible provided that there is financial support to cover bioresource costs not paid by charge back fees. Alternatively, some bioresources can compensate for this financial deficit by distributing extensive numbers of biospecimens to companies at higher fees.

Commercial companies frequently require human tissues to support their research and product development. It is important to provide such companies with human biospecimens, as they play an important role in the development of therapeutic approaches to a wide range of diseases. Typically, many commercial companies use biospecimens that are not frequently requested by academic investigators, particularly ‘normal’ tissues. To ensure that a commercial company ‘pays its way’, the charge back cost is, as a minimum, the total cost of collecting, processing and distributing each biospecimen. This currently stands at usually about US$200 or more.

What investigators need to know about using human tissues in their research

If an investigator intends to replace some of the animals that they currently use in research with human tissues, then the approach should be carefully considered and planned. Investigators transitioning from animals to human tissues should consider performing a pilot study to test their approach on a small number of human biospecimens. Although human tissues may be readily available, bioresources are often reluctant to provide large numbers of biospecimens until such a small-scale pilot study has demonstrated that the approach is promising. For example, in studies on common tumours, a pilot study should typically be based on around 5–10 biospecimens. After the experimental results are analysed and shown to be encouraging, additional tumour specimens may be requested, along with an experimental plan to confirm and expand the initial results.

The availability of tissue from bioresources can be limited if requests are too restrictive. In general, the actual request for a tissue determines its availability. For example, solid tissues may be difficult to obtain if, for example, the request is for:

— tissues from patients whose diseases are not treated by surgery (e.g. type-1 diabetes);

Because of these issues, it is highly recommended that investigators needing biospecimens from a bioresource discuss their needs with staff from the bioresource early in the project planning process. Staff from the bioresource can help investigators refine their requests for biospecimens and help them through the application process. If a bioresource is unable to fill the investigator’s request, the staff can sometimes refer them to other more appropriate bioresources.

Bioresources undertake a number of approaches to maximise the requests that can be filled from their inventories. For example, to increase the tissues available to support research, solid biospecimens are divided into standard size aliquots during processing. The paraffin blocks used for QC evaluation (see later section) represent other samples which can also be used for distribution purposes. Similarly, plasma, serum, buffy coats and other bodily fluids are aliquoted into standard volumes.

Safety issues

While care must be taken when working with animals and cell lines, research with human-derived solid tissues and bodily fluids requires more attention to safety because some human tissues may be infected with human pathogens. Usually, cell lines have been screened for common human pathogens; however, this may not be the case for human solid tissues and bodily fluids, and thus universal precautions for working with potentially biohazardous materials are required. ⁴³ Investigators should follow their institution’s safety plans/guidelines for the handling and disposal of biohazardous materials. These plans may require the use of protective safety equipment, including appropriate gloves, masks, safety glasses and protective coverings (such as aprons), to prevent exposure of the skin and clothes to human tissue products.

Variables affecting the use of solid human tissues in research

When working with human tissues, investigators need to be aware of the variables that might affect their research. Human tissues, like the individuals from whom they are obtained, are quite variable; donors vary with respect to age, race, sex, ethnicity and many other factors. From the standpoint of surgically removed human tissues, additional variables can be classified in terms of the stage at which the tissue was resected. For example, tissues may be removed before diagnosis, as part of diagnosis, or after diagnosis. They may also be obtained before, during and after neoadjuvant therapy and before, during, or after definitive surgery.

Variables also may occur during collection, processing, storage and distribution of biospecimens. While many variables exist at this stage, they may not all be equally important — nonetheless, they are variables of which investigators must be aware, in order to avoid bias in the interpretation of their data.

Pre-diagnostic variables are numerous. The most important are probably comorbidities, which are diseases present in tissue donors prior to diagnosis of the operative disease. The more common comorbidities include: cancer; type 1 and 2 diabetes; hypertension; renal failure; and liver failure. Other pre-diagnostic variables are diet, and environmental and occupational exposures. More detailed information on all variables has been described previously. ⁴⁴

Diagnostic and post-diagnostic variables usually are related to the tissues obtained after diagnostic biopsy, analysis of biofluids or imaging. When a biopsy is obtained, tissues surrounding the biopsy site are frequently affected by trauma, tissue damage and associated changes such as scarring and inflammation. Human tissues used in research can sometimes include an uncharacterised portion of a biopsy site, which can confound the interpretation of the research findings.

Neoadjuvant therapy is therapy which is given to a potential tissue donor prior to definitive surgery. This includes chemotherapy, biologic therapy (such as Herceptin®) and/or radiation therapy. Currently, tissue effects, especially the molecular changes caused by neoadjuvant therapy, are generally unknown, so tissues obtained after neoadjuvant therapy may not be useful for research purposes and probably should be avoided.

Variables associated with the definitive surgery include anaesthesia, cauterisation and other damage to tissues, and the duration of warm ischaemia. Cauterisation in surgery is preferred by many surgeons because it coagulates blood vessels, thus reducing blood in the surgical field and overall blood loss. Investigators should be aware that, during cauterisation, heat can spread superficially from the site of the surgical cut into some adjacent tissues. This associated temperature increase in the tissue can affect immunohistochemistry and other molecular assays.

Warm ischaemia begins when the tissue is partially or completely isolated from the vascular supply, and ends when the tissue is removed from the patient’s body. During this ‘warm ischaemic’ period, the tissue experiences low oxygen levels, but at approximately body temperature. This means that the cellular enzymes will still be functional, and thus have the potential to cause cellular damage. ‘Cold ischaemia’ begins when the tissue is removed from the body and ends when tissue stabilisation by fixation or freezing begins. Significant ischaemic damage can occur during both warm and ‘early’ cold ischaemia (e.g. 10 minutes after surgical removal from the patient).^45,46

Processing the remnant tissue (i.e. tissue no longer needed for clinical care) provided by surgical pathology for research purposes typically includes portioning and preparing the aliquots for distribution or for storage. Storage preparation may include fixing the tissue in neutral buffered formalin and embedding it in paraffin blocks (FFPE), or preparing aliquots for freezing and storage at ultracold temperatures.

The type of biospecimens must fit the type of analyses that will be performed on them (i.e. they should be ‘fit-for-purpose’). Some assays are best performed on frozen biospecimens, while others can use FFPE samples. ⁴⁷ It is noteworthy that, compared to frozen tissue, extraction of mRNA from FFPE is not as efficient. Therefore, a larger FFPE biospecimen would be required to extract mRNA. Whether frozen or FFPE samples are most appropriate for the research depends upon the research questions being asked, the specific cellular or molecular targets, and the types of analyses that will be performed. Based on these considerations, investigators must decide which assays and biospecimens (including size) are most suitable for their work.

While the pre-analytical variables inherent in human tissues cannot be controlled as rigorously as those in experimental animals (e.g. by strictly monitoring of their diet), there are significant quality control measures that are practised by bioresources that provide high-quality human biospecimens for research. These include: ensuring that the biospecimens are processed rapidly; minimising collection and processing variables; and recording any such pre-analytical variables to help document their impact. ⁴⁴ For example, most bioresources maintain records on the duration of warm and cold ischaemia, and follow SOPs to limit tissue fixation variables.^32,47 Bioresources that follow best practices take precautions to avoid potentially detrimental extended incubations in formalin — for example, by avoiding weekend tissue processing runs.

In addition, information is documented beyond that recorded by the pathologist. For example, in the Cooperative Human Tissue Network (CHTN), QC procedures include providing a separate diagnostic description of each biospecimen distributed to an investigator. For QC evaluation of each aliquot in the bank, a matching portion of tissue is processed to FFPE. For cancer-related biospecimens, this evaluation includes determining the percentage of cancer in the biospecimen and, within the cancerous tissue itself, the percentage of malignant nuclei, the extent of necrosis, and the percentage of stroma, fat and inflammatory cells. ³² This information is provided for each aliquot distributed to an investigator.

In summary, bioresources that follow best practices collect and process tissues according to well-established SOPs. They also routinely maintain records on the duration of warm and cold ischaemia, tissue fixation (including fixative and fixation times), and any other variables related to tissue processing. Annotation of data on the tissue source includes the donor’s age, race, sex and information from their electronic medical records, where needed. ²²

Finally, although bioresources follow a range of well-established SOPs to provide high-quality biospecimens, bioresources and/or researchers, when needed, can also perform QC assays such as RIN, DV200, delta Cq qRT-PCR and mass spectrometry-based assays in order to identify biospecimens that might be compromised in quality for various analytes. However, such assays can be expensive to perform.

Variables affecting the use of human bodily fluids in research

The key variables to consider when using human bodily fluids are: collection; processing; storage and distribution. Bodily fluids are best collected prospectively from consenting patients, and processed rapidly. Blood may be collected at clinic/office visits when the blood is not needed from a fasting individual, or before surgery when the patient is fasting. Disparate samples and other assay components should not be mixed or analysed in the same assay; this includes mixing fasting and non-fasting blood, serum and plasma, and different sample containers.

In the collection of blood and blood products, haemolysis should be avoided by the choice of needles and control of the rate of withdrawal. In addition, when storing or transporting whole blood on solid ice, care must be taken to avoid freezing the sample to minimise haemolysis. ⁴⁸ ‘Normal’ blood depends upon the characteristics of the individual from whom the blood sample is collected — specifically, blood from a cancer patient would not be considered to be ‘normal’. Normal blood is best obtained prospectively from volunteer donors without known major diseases.

Summary and conclusions

Animals serve as important resources for scientific research. However, research findings in animals often must be confirmed using human tissues. As we have discussed in this paper, the use of human tissues has led to major advances in science, technology and medical care. While numerous sources of human tissues exist, researchers making the transition from research using animals to human tissues may not be aware of how to identify sources of high-quality biospecimens and use them effectively in their research.

Many models of bioresources and sources of high-quality biospecimens exist, and some may be more appropriate than others for certain types of research. A number of tools are available to help researchers find the most appropriate sources of high-quality biospecimens for their work. Once potential sources of human biospecimens have been identified, it is essential for researchers to discuss their needs with bioresource personnel who can help them refine their requests or seek other, more appropriate, bioresources. It is critical that the type of biospecimens requested are ‘fit-for-purpose’ for the research questions and analyses to be performed.

In order to ensure that the findings obtained from the use of human tissues are accurate, generalisable and free from bias, it is important for researchers to understand the variables that can affect the use of human tissues and the impact these variables can have on their results. Careful consideration of these and other issues highlighted in this paper can help researchers transition effectively from research involving the use of animals to research on human tissues and contribute to scientific and medical advancements.