HT22 is a murine neuronal cell line commonly employed in neuroscience research. It is valuable for studying neurogenerative disorders and testing potential neuroprotective therapies or treatments. Besides, the HT22 cells are glutamate-sensitive thus, are used to investigate glutamate-induced toxicity or neuronal damage.

This article highlights the imperative aspects of HT22 cells that can assist you in your research work. It will mainly discuss:

  1. General attributes and origin of HT22 cells
  2. HT22 cell line culturing information
  3. Advantages & disadvantages of HT22 cell line
  4. Research applications of HT22 cell line
  5. Research publications featuring HT22 cells
  6. Resources for HT22 Cells: Protocols, Videos, and More

Amyloid Plaques forming between neurons in Alzheimer's disease.

1.      General attributes and origin of HT22 cells

This section of the article will shed light on the origin and general characteristics of HT22 cells. Knowing this information about a cell line is necessary before commencing work on it. Herein, you will learn: What are HT 22 cells? What is the morphology of HT22 hippocampal cells? What is HT22 cell size?

HT22 is a continuous cell line derived from the hippocampus region of the mouse brain. It is a subclone of the parental HT-4 cell line developed by immortalization of murine neuronal tissues via a temperature-sensitive polyomavirus simian virus 40 (SV40) T antigen.

These cells differ from mature hippocampal cells as they lack glutamate and cholinergic receptors like mature neurons of hippocampal origin. Thus, they are not suitable for memory-related research studies [1].

HT22 cells have an epithelial cell-like appearance.

2.      HT22 cell line culturing information

HT22 cells are used in neuroscience research laboratories. Before culturing these cells, researchers look for essential cell culture information that makes their work easy and efficient. This section covers all the key points for culturing HT22 cells. You will know: What is the HT22 cell doubling time? How do you culture HT22 cells? What is the HT22 cell culture protocol? What is the HT22 cell media?

Key Points for Culturing HT22 Cells

Doubling Time:

The HT22 cell doubling time is approximately 15 hours.

Adherent or in Suspension:

HT22 cells are adherent.

Sub-cultivation Ratio:

The sub-cultivation ratio for HT22 hippocampal cells is 1:3 to 1:6. Briefly, media is removed, and adherent cells are rinsed with 1 x PBS. Accutase dissociating solution is added into the flask, and cells are incubated for 8 to 10 minutes at an ambient temperature. Afterwards, fresh culture media is added, and cells are collected in a vial for centrifugation. The obtained cell pellet is carefully resuspended, and cells are dispensed into a culture flask for growth.

Growth Medium:

DMEM media is used for culturing HT22 cells. It is supplemented with 10% FBS, 4.5 g/L Glucose, 4 mM L-Glutamine, 1.5 g/L NaHCO3, and 1.0 mM Sodium pyruvate for optimum cell growth.

Growth Conditions:

HT22 cell cultures are kept in a humidified incubator (37 °C temperature) with a 5% CO2 supply.


Frozen HT22 cells can be stored at below -150 °C temperature either in the vapour phase of liquid nitrogen or in an ultra-low temperature electric freezer for the long term.

Freezing Process and Medium:

HT22 cells can be frozen in CM-1 or CM-ACF media using a slow freezing method. This process allows only a 1 °C decrease in the sample’s temperature per minute, protects cells from shock, and helps maintain their viability.

Thawing Process:

Cells are thawed in a 37 °C water bath for 40 to 60 seconds until a small ice clump is left. Afterwards, culture media is added into cells, and they are centrifuged to eliminate freezing media components. The cell pellet is resuspended and cells are poured into the new flask containing culture media. Following this, cells are incubated at 37 °C in an incubator for at least 24 hours.

Biosafety Level:

Biosafety level 1 laboratory settings are mandatory to culture HT22 cells.


30% and 90% confluent monolayers of HT22 cells in cell culture.

3.      Advantages & disadvantages of HT22 cell line

HT22 hippocampal cells are associated with some pros and cons that differentiate them from other neuronal cell lines. Some notable advantages and disadvantages of the cell line are mentioned here.


The advantages of the HT22 murine neuronal cell line are:

Fast Growth Rate

HT22 cells have a doubling time of 15 hours, enabling rapid and efficient experimentation and aiding in timely research outcomes.


HT22 is an immortalized cell line, ensuring continuous growth for extended periods. This provides consistent availability of cells, reduces time and cost, and facilitates long-term experiments with consistent results.



The disadvantages of HT22 cells are:

Murine Origin

The HT22 cell line was derived from mouse brain hippocampus tissue, which may not fully represent the complexity of human neuron cell physiology and behaviour, limiting their translational relevance.


4.      Research applications of HT22 cell line

The HT22 cells are extensively used in neurobiology research. A few promising applications of this cell line are discussed in this section of the article:

  • Neuroscience research: HT22 cells are widely employed in neurodegenerative disease research, i.e., Alzheimer’s and Parkinson’s disease. They are considered a valuable research tool for studying neurotoxicity and oxidative stress mechanisms related to these diseases. Research conducted in 2020 found that the PI3K/AKT/CREB pathway is involved in hyperglycemia-induced neural toxicity in HT22 cells [2]. Similarly, a recent study proposed that the Nrf2/HO‐1 pathway and NF- κB signalling axis play significant roles in HT22 amyloid beta toxicity [3].
  • Drug screening: HT22 cells are broadly used for drug testing and screening purposes. They help researchers identify potential therapeutic agents exhibiting neuroprotective effects for combating neurodegenerative diseases. A study conducted in 2019 explored the neuroprotective potential of tetrahydro curcumin compound in glutamate-treated HT22 hippocampal cells. Herein, glutamate induces oxidative stress in HT22 cells and causes cell death by activating mitogen-activated protein kinases [4].

5.      Research publications featuring HT22 cells

Here are some interesting research papers featuring the HT22 cell line:

Investigation of the neuroprotective effects of crocin via antioxidant activities in HT22 cells and in mice with Alzheimer’s disease

This study in the International Journal of Molecular Medicine (2019) proposed the neuroprotective potential of crocin, a compound isolated from the Crocus sativus L. in the Lglutamatedamaged HT22 cells.

Novel oxindole derivatives prevent oxidative stress-induced cell death in mouse hippocampal HT22 cells

This publication in Neuropharmacology (2018) reported that oxindole derivatives protect against HT22 cell death induced by oxidative stress.

Ginsenoside Rb2 suppresses the glutamate-mediated oxidative stress and neuronal cell death in HT22 cells

This research paper was published in the Journal of Ginseng Research in 2019. This study explored the neuroprotective effects of a natural product, ginsenoside Rb2, using HT22 cell line. The study found that ginsenoside rb2 efficiently reduced glutamate-induced oxidative stress and cell death in murine hippocampal cells HT22.

Silver nanoparticles induced cytotoxicity in HT22 cells through autophagy and apoptosis via PI3K/AKT/mTOR signaling pathway

This study in Ecotoxicology and Environmental Safety (2021) evaluated the cytotoxic potential of silver nanoparticles in the HT22 cell line.

Ferrostatin-1 protects HT-22 cells from oxidative toxicity

This research article was published in 2020 in the Neural regeneration research. It proposed that ferrostatin-1, an inhibitor of ferroptosis, prevents oxidative toxicity in HT22 hippocampal cells.

6.      Resources for HT22 Cells: Protocols, Videos, and More

Some online resources on the HT22 cells explain their transfection, differentiation, and cell culture protocols:

  • HT22 transfection: This document contains an optimized protocol for HT22 transfection in 24 and 96-well cell culture plates.
  • HT22 cell differentiation: This article will comprehensively guide you about the HT22 differentiation protocol.

The following link contains the HT22 cell culture protocol:

  • HT22 cell subculturing: This link will help you learn the sub-cultivation protocol for the HT22 cell line. Moreover, it will assist you in learning the protocol for inducing neurotoxicity in cells.
  • HT22 hippocampal cells: This website contains a lot of useful information about HT22 cell doubling time, media, and cell culture protocols.


  1. He, M., et al., Differentiation renders susceptibility to excitotoxicity in HT22 neurons. Neural Regen Res, 2013. 8(14): p. 1297-306.
  2. Zhang, S., et al., Fisetin prevents HT22 cells from high glucose-induced neurotoxicity via PI3K/Akt/CREB signaling pathway. Frontiers in Neuroscience, 2020. 14: p. 241.
  3. Zhang, R.-l., et al., Protective effects of berberine against β-amyloid-induced neurotoxicity in HT22 cells via the Nrf2/HO‐1 pathway. Bioorganic Chemistry, 2023. 133: p. 106210.
  4. Park, C.-H., et al., Neuroprotective effects of tetrahydrocurcumin against glutamate-induced oxidative stress in hippocampal HT22 cells. Molecules, 2019. 25(1): p. 144.